Product Search for IgG: Novus Biologicals

AB-108-C

Goat IgG Isotype Control [Unconjugated]

(2 Reviews)

1 mg

AB-108-C

AF496, filled histogram) or Normal Goat IgG Isotype Control Antibody (Catalog # AB-108-C, open histogram), followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107)." class="big_lightbox">

SH-SY5Y human neuroblastoma cell line was stained with Goat Anti-Human/Mouse/Rat Ephrin-B2 Antigen Affinity-purified Polyclonal Antibody (Catalog # <a class=

AF496, filled histogram) or Normal Goat IgG Isotype Control Antibody (Catalog # AB-108-C, open histogram), followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107)." alt="SH-SY5Y human neuroblastoma cell line was stained with Goat Anti-Human/Mouse/Rat Ephrin-B2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF496, filled histogram) or Normal Goat IgG Isotype Control Antibody (Catalog # AB-108-C, open histogram), followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107)." class="big_thumb" />

<b>Biological Strategies Validation. </b>Decreased macrophage recruitment correlates with decreased angiogenesis.A) Quantification of BrdU incorporation demonstrates that decreased macrophage infiltration does not significantly correlate with a change in epithelial cell proliferation (N.S. = not significant). B) Quantification of VWF staining reveals decreased blood vessels associated with epithelial structures in mammary glands from mice treated with B/B and anti-CX3CR1 blocking antibody compared with mice treated with B/B and IgG control antibody. *p<0.05. C, D) Representative images of VWF stained mammary glands from mice treated with B/B and either control IgG antibody (C) or anti-CX3CR1 (D). Green = VWF staining, blue = DAPI. Arrows indicate VWF-positive structures. Scale bars represent 50 µM. Results in each figure panel are representative of a minimum of three different mice for each treatment group and genotype. Image collected and cropped by CiteAb from the following open publication (//dx.plos.org/10.1371/journal.pone.0045877), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>iFGFR1 activation in vivo promotes recruitment of CX3CR1-positive macrophages.MMTV-iFGFR1 transgenic mice were treated with B/B in order to analyze the population of macrophages that are recruited to the mammary epithelium during early stages of iFGFR1-induced mammary tumorigenesis. A) MMTV-iFGFR1 mice treated with B/B demonstrated an increase in macrophage recruitment after 10 days as indicated by an increased in the number of F4/80 positive cells. MMTV-iFGFR1 mice treated with anti-CX3CR1 in conjunction with B/B demonstrated a reduction in macrophage recruitment at 10 days indicating that iFGFR1 activation is responsible for recruiting a subset of macrophages that express CX3CR1. ***p<0.0001. Error bars represent SEM. B) Representative image of macrophages associated with budding epithelial structures in mammary glands from mice treated with control IgG antibody. C) Representative image of macrophages associated with budding epithelial structures in mammary glands from mice treated with anti-CX3CR1. Red = F4/80 staining, blue = DAPI. Scale bars represent 50 µM. Results in each figure panel are representative of a minimum of three different mice for each treatment group and genotype. Image collected and cropped by CiteAb from the following open publication (//dx.plos.org/10.1371/journal.pone.0045877), licensed under a CC-BY license. Not internally tested by R&D Systems.

Blocking the tissue inhibitor of metalloproteinase type 1 (TIMP-1) in MSC-CM reduced its pro-oligodendroglial activity on cultured aNSCs. Representative immunocytochemical staining of 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase)- (A) and GFAP-positive (B) cells treated either with control (no antibody), anti-TIMP-1, or immunoglobulin G (IgG) control pre-incubated with alpha -MEM or MSC-CM after 3 days in culture. (C) Quantification of CNPase- and GFAP-positive (D) cell numbers. Number of experiments n = 4. Statistical significance was calculated using a two-way ANOVA with Bonferroni posttest: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32570968), licensed under a CC-BY license. Not internally tested by R&D Systems.

Localisation of Sox17 staining in proliferative and secretory phase endometrial tissue and regulation by hormones. Within the proliferative (A) and secretory (B) endometrium, Sox17 localized to the glandular (open arrowheads) and luminal (closed arrowheads) epithelium, with staining appearing in an irregular, patchy, pattern. Smaller inset boxes show relevant IgG negative controls. Imaged at 20x magnification, scale bar = 50 μm. Treatment of endometrial luminal epithelial (ECC-1) cells with 10−8 M estrogen (estradiol)/10−7 M progesterone (medroxyprogesterone acetate) (, *p < 0.05) resulted in an upregulation of Sox17 protein, when compared to untreated, estradiol only (■, E) treatment groups. (C,D) Data presented as mean ± SEM, n = 5. Representative Western immunoblot shown. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31664088), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Genetic Strategies Validation. </b>Knockdown of SOX17 results in reduced expression in endometrial luminal epithelial cells. Transfection of ECC-1 cells with a CRISPR/Cas9 SOX17 knock down plasmid resulted in decreased SOX17 expression (KD1-5) when compared to untransfected control ECC-1 cells (ECC-1) and ECC-1 cells transfected with a control plasmid (CP). Analysed by Western immunoblot, representative blot shown. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31664088), licensed under a CC-BY license. Not internally tested by R&D Systems.

Osteoclast resorption of bony interspinous ligaments releases active TGF-beta to drive the progression of ossification in AS patients. e Immunostaining and f quantitative analysis of CD68-positive cells (brown) in the normal and HO-formed interspinous ligaments (sagittal view). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panel); 25 μm (bottom panel). Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33731675), licensed under a CC-BY license. Not internally tested by R&D Systems.

Progression of endochondral heterotopic ossification in AS patients. c Immunostaining and d quantitative analysis of collagen II-positive cells (brown) in the normal and chondrogenic interspinous ligaments (sagittal view). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panels); 25 μm (bottom panels). Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33731675), licensed under a CC-BY license. Not internally tested by R&D Systems.

Chondrocyte differentiation and cartilage formation in interspinous ligaments of AS patients before calcification. a Hematoxylin and eosin (H&E) staining and b Safranin O–Fast Green (SOFG) staining of normal and chondrogenic interspinous ligaments. In the AS group, the right panels show magnified views of the boxed area in the left panels. Scale bar: 100 μm (two panels on the right side); 25 μm (left panel). c Immunostaining and d quantitative analysis of collagen II-positive cells (brown) in the normal and chondrogenic interspinous ligaments (sagittal view). The bottom panels show a magnified view of the boxed area in the top panels. Scale bar: 100 μm (top panel); 25 μm (bottom panel). e Immunostaining and f quantitative analysis of pSmad2/3-positive cells (brown) in the normal and chondrogenic interspinous ligaments (sagittal view). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panel); 25 μm (bottom panel). g Immunostaining and h quantitative analysis of pSmad1/5/8-positive cells (brown) in the normal and inflammatory interspinous ligaments (sagittal view). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panels); 25 μm (bottom panels). C, cartilage; L, ligament Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33731675), licensed under a CC-BY license. Not internally tested by R&D Systems.

Progression of endochondral heterotopic ossification in AS patients. i Immunostaining and j quantitative analysis of pSmad2/3-positive cells (brown) in the normal ligaments and endochondral-ossified interspinous ligaments (sagittal view). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panel); 25 μm (bottom panel). k Immunostaining and l quantitative analysis of Osterix-positive cells (brown) in the normal and endochondral-ossified interspinous ligaments (sagittal view). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panel); 25 μm (bottom panel). m Immunostaining and n quantitative analysis of pSmad1/5/8-positive cells (brown) in the normal and inflammatory interspinous ligaments (sagittal view). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panels); 25 μm (bottom panels). B, bone; BM, bone marrow; C, cartilage Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33731675), licensed under a CC-BY license. Not internally tested by R&D Systems.

Elevated TGF-beta levels in the early inflammatory stage of AS. a Hematoxylin and eosin (H&E) staining and b Safranin O–Fast Green (SOFG) staining of normal and inflamed interspinous ligaments. In the AS group, the right panels show magnified views of the boxed area in the left panels. Scale bar: 100 μm (two panels on the right); 25 μm (left panel). c Immunostaining and d quantitative analysis of CD68-positive cells (brown) in the normal and inflamed interspinous ligaments (sagittal view). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panels); 25 μm (bottom panels). e Immunostaining and f quantitative analysis of pSmad2/3-positive cells (brown) in the normal and inflamed interspinous ligaments (sagittal view) in normal ligaments and inflammatory ligaments. The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panels); 25 μm (bottom panels). g Immunostaining and h quantitative analysis of pSmad1/5/8-positive cells (brown) in the normal and inflamed interspinous ligaments (sagittal view). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panels); 25 μm (bottom panels) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33731675), licensed under a CC-BY license. Not internally tested by R&D Systems.

Osteoclast resorption of bony interspinous ligaments releases active TGF-beta to drive the progression of ossification in AS patients. g Immunostaining and h quantitative analysis of pSmad2/3-positive cells in the normal and HO-formed interspinous ligaments (sagittal view). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panel); 25 μm (bottom panel). i Immunostaining and j quantitative analysis of Osterix-positive cells (brown) in the normal and HO-formed interspinous ligaments (sagittal view). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panel); 25 μm (bottom panel).Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33731675), licensed under a CC-BY license. Not internally tested by R&D Systems.

Progression of endochondral heterotopic ossification in AS patients.g Immunostaining and h quantitative analysis of the number of CD68-positive osteoclast (brown) surface (OCS) per bone surface (BS). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panel); 25 μm (bottom panel). i Immunostaining and j quantitative analysis of pSmad2/3-positive cells (brown) in the normal ligaments and endochondral-ossified interspinous ligaments (sagittal view). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panel); 25 μm (bottom panel). k Immunostaining and l quantitative analysis of Osterix-positive cells (brown) in the normal and endochondral-ossified interspinous ligaments (sagittal view). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panel); 25 μm (bottom panel). m Immunostaining and n quantitative analysis of pSmad1/5/8-positive cells (brown) in the normal and inflammatory interspinous ligaments (sagittal view). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panels); 25 μm (bottom panels). B, bone; BM, bone marrow; C, cartilage Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33731675), licensed under a CC-BY license. Not internally tested by R&D Systems.

Increased LCN2 expression in Esr1-deficient ovaries. Ovaries from wild-type (WT, n = 8) and Esr1 null mice (n = 6) were dissected and either embedded in paraffin or prepared for RNA analysis. (A) Hematoxylin and eosin (H & E) staining showed characteristic histological changes in Esr1-deficient compared to WT ovaries. (B) Immunohistochemical staining for LCN2 and ER alpha was performed. Sections were counterstained with hematoxylin (LCN2) or methyl green (ER alpha ). Magnifications: 100×, 400×; scale bars: 100 µm, 50 µm. (C) Tissue samples were subjected to immunofluorescence double staining for LCN2 and STAR. Magnifications: 100×; scale bars: 100 µm. (D) Lcn2 and Star expression was analyzed in ovarian tissues using RT-qPCR. Data are displayed as means ± SD. In case of normal distribution, Student’s t-tests were chosen for statistical analysis, otherwise a Mann–Whitney test was performed. Significant differences between groups are marked with asterisks: *** p < 0.001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37298232), licensed under a CC-BY license. Not internally tested by R&D Systems.

Reactivity:

Gt

Species Glossary

Reactivity: Gt

Applications:

Applications:

Host:

Goat Polyclonal

Host: Goat Polyclonal

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 488	IC108G
Alexa Fluor 647	IC108R
Alexa Fluor 700	IC108N
Allophycocyanin	IC108A
Allophycocyanin	AB-108-CA
Biotin	AB-108-CB
Biotin	BAF108
DyLight 350	AB-108-CD
DyLight 405	AB-108-CE
DyLight 488	AB-108-CK
DyLight 550	AB-108-CL
DyLight 594	AB-108-CM
DyLight 650	AB-108-CW
DyLight 680	AB-108-CY
DyLight 755	AB-108-CZ
FITC	AB-108-CF
Fluorescein	IC108F
HRP	AB-108-CH
Janelia Fluor 525	AB-108-CJF525
Janelia Fluor 549	AB-108-CI
Janelia Fluor 646	AB-108-CJ
Janelia Fluor 669	AB-108-CJF669
PE	AB-108-CP
PerCP	AB-108-CC
PerCP (Peridinin-chlorophyll Protein Complex)	IC108C
Phycoerythrin	IC108P

Available Formulations

Formulation	Catalog #	Availability	Price
Carrier Free	IC108A
Carrier Free	IC108C
Carrier Free	IC108F
Carrier Free	IC108G
Carrier Free	IC108N
Carrier Free	IC108P
Carrier Free	IC108R

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AF-401-NA

IL-1 beta/IL-1F2 Antibody [Unconjugated]

(3 Reviews)

(610 Publications)

100 μg

AF-401-NA

HAF017). A specific band was detected for IL-1 beta /IL-1F2 at approximately 35 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1." class="big_lightbox">

<b>Biological Strategies Validation. </b>Western blot shows lysates of THP-1 human acute monocytic leukemia cell line untreated (-) or treated (+) with 200 nM PMA for 24 hours and 10 µg/mL LPS for 4 hours and RAW 264.7 mouse monocyte/macrophage cell line untreated (-) or treated (+) with 10 µg/mL LPS for 24 hours. PVDF membrane was probed with 0.25 µg/mL of Goat Anti-Mouse IL-1 beta /IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (<a class=

HAF017). A specific band was detected for IL-1 beta /IL-1F2 at approximately 35 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1." alt="Western blot shows lysates of THP-1 human acute monocytic leukemia cell line untreated (-) or treated (+) with 200 nM PMA for 24 hours and 10 µg/mL LPS for 4 hours and RAW 264.7 mouse monocyte/macrophage cell line untreated (-) or treated (+) with 10 µg/mL LPS for 24 hours. PVDF membrane was probed with 0.25 µg/mL of Goat Anti-Mouse IL-1 beta /IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF017). A specific band was detected for IL-1 beta /IL-1F2 at approximately 35 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1." class="big_thumb" />

NL001) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips." class="big_lightbox">

<b>Biological Strategies Validation. </b>IL-1 beta /IL-1F2 was detected in immersion fixed MCF-7 human breast cancer cell line using Goat Anti-Mouse IL-1 beta /IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) at 8 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; <a class=

NL001) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips." class="big_thumb" />

CTS008) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Frozen Tissue Sections." class="big_lightbox">

IL-1 beta /IL-1F2 was detected in perfusion fixed frozen sections of mouse thymus using Goat Anti-Mouse IL-1 beta /IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) at 15 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; <a class=

CTS008) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Frozen Tissue Sections." class="big_thumb" />

HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system." class="big_lightbox">

<b>Biological Strategies Validation. </b>Simple Western lane view shows lysates of RAW 264.7 mouse monocyte/macrophage cell line untreated (-) or treated (+) with 10 µg/mL LPS for 24 hours, loaded at 0.5 mg/mL. A specific band was detected for IL-1 beta /IL-1F2 at approximately 40 kDa (as indicated) using 2.5 µg/mL of Goat Anti-Mouse IL-1 beta /IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (<a class=

HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system." class="big_thumb" />

401-ML) stimulates proliferation in the the D10.G4.1 mouse helper T cell line in a dose-dependent manner (orange line) as measured by Resazurin (AR002). Proliferation elicited by Recombinant Mouse IL-1 beta /IL-1F2 (50 pg/mL) is neutralized (green line) by increasing concentrations of Goat Anti-Mouse IL-1 beta /IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA). The ND₅₀ is typically ≤ 0.25 µg/mL." class="big_lightbox">

Recombinant Mouse IL-1 beta /IL-1F2 (<a class=

401-ML) stimulates proliferation in the the D10.G4.1 mouse helper T cell line in a dose-dependent manner (orange line) as measured by Resazurin (AR002). Proliferation elicited by Recombinant Mouse IL-1 beta /IL-1F2 (50 pg/mL) is neutralized (green line) by increasing concentrations of Goat Anti-Mouse IL-1 beta /IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA). The ND₅₀ is typically ≤ 0.25 µg/mL." class="big_thumb" />

VC006). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in epithelial cells. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents." class="big_lightbox">

IL‑1 beta /IL‑1F2 was detected in perfusion fixed paraffin-embedded sections of Mouse Intestine using Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) at 5 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Sheep IgG VisUCyte™ HRP Polymer Antibody (<a class=

VC006). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in epithelial cells. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents." class="big_thumb" />

<b>Biological Strategies Validation. </b>Quercetin inhibits auto-reactive NLRP3 inflammasome.(A) IL-1 beta concentrations in Nlrp3A350V/A350V and WT BMDM culture supernatants primed with LPS (500 ng/ml) for 90 min and treated with quercetin 1 h after LPS. (B) Supernatants of BMDM were analyzed by anti-ASC, anti-Caspase-1 immunoblotting. Lysates of BMDM were analyzed by anti-IL-1 beta and anti-beta -actin immunoblotting. Data shown are representative of two or more experiments (means ± SD) ***p < 0.001. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28148962), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>GRA7-I-induced inflammasome activation was required for antimicrobial activity in MTB-infected macrophages.(A and B) BMDMs from PKC alpha +/+ and PKC alpha -/- mice (A) and BMDMs were transduced with lentivirus-shRNA-NS or lentivirus-shRNA-ASC for 2 days (B) infected with MTB (MOI = 1) for 4 h and then stimulated with rGRA7 (1, 5, 10 μg/ml) and its mutants for 18 h. IB analysis for IL-1 beta p17, IL-18 p18, or caspase-1 p10 in supernatants (SN), ASC, NLRP3, pro-IL-1 beta , pro-IL-18, or pro-caspase-1 in whole-cell lysates (WCL). Actin was used as a loading control. (C and D) Intracellular survival of MTB was assessed by CFU assay. BMDMs were infected with MTB for 4 h, followed by treatment with rGRA7, and then lysed to determine intracellular bacterial loads. The data are representative of five independent experiments with similar results (A and B). Data shown are the mean ± SD of five experiments (C and D). Significant differences (*P < 0.05; **P < 0.01; ***P < 0.001) compared with rVector. CFU, colony-forming units. ns, not significant. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1006126), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>M. bovis-induced inflammasome activation requires ERS. (A) Immunoblot analysis at different time-points of Bip and p-IRE1 alpha in lysates of BMDMs infected with M. bovis (MOI 10). (B) Immunoblot analysis at different time-points of IL-1 beta in supernatants of BMDMs infected with M. bovis (MOI 10). (C) ELISA for IL-1 beta analysis from supernatants of BMDMs infected with M. bovis (MOI 10). (D) IL-1 beta immunoblot analysis of supernatants from BMDM infected for 24 h with M. bovis (MOI 10) in the presence or absence of 4-PBA. (E) ELISA for IL-1 beta quantification from supernatants of BMDMs treated with or without 4-PBA for 1 h and then infected with M. bovis (MOI 10) for 24 h. ELISA for (F) TNF- alpha and (G) IL-6 detection in supernatants from M. bovis (MOI 10) infected BMDMs. ELISA for (H) TNF-alpha and (I) IL-6 detection from supernatants of BMDMs treated with or without 4-PBA for 1 h and then infected with M. bovis (MOI 10) for 24 h. TM, tunicamycin, positive control for ERS induced mitochondrial damage, 10 μg/mL; LPS+ATP, positive control for inflammasome activation, 200 ng/mL, and 1 mM, respectively; LPS, positive control for TNF-alpha and IL-6 production, 200 ng/mL; UNT, untreated; 4-PBA, 4-phenyl butyric acid, ERS inhibitor, 5 mM; MOI, multiplicity of infection. For (C,F,G), Data are representative of at least three independent experiments, each performed in triplicate for WB. The results are shown are the mean ± SD. The asterisks indicate statistically significant differences compared with untreated cells (*P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant). P-values were obtained by using one-way ANOVA followed by post-hoc Turkey' comparison test. For (E,H,I), data are representative of at least three independent experiments. The results are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. P-values were analyzed using Student's t-test. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30846986)

<b>Biological Strategies Validation. </b>NLRP1A is activated by N-terminal proteolysis.(A) The amino acid sequence of the first 244 residues of NLRP1AB6 was aligned to NLRP1B129 and NLRP1BB6. The arrow above the alignment indicates the LF-cleavage site in NLRP1B129. (B) 293T cells were transfected for 36h with 200ng empty vector (V) or plasmids pcDNA3.1-HA-NLRP1B129 or -NLRP1AB6-MYC (CMV promoter) along with 200ng mCASP1 and 200ng mIL-1 beta expression vectors. Cells were treated overnight with anthrax lethal toxin (LeTx, 1μg/ml) 24h post-transfection, and then lysates were analyzed by immunoblotting (IB) with the indicated antibodies. (C) 293T cells were transfected for 36h with plasmids encoding 400ng GFP-HA-NLRP1B129 or GFP-HA-NLRP1AB6 (under the control of the LTR promoter in pMSCV) or with mutants engineered to contain an N-terminal TEV protease site. Cells were also co-transfected with 100ng empty vector (V) or plasmids encoding TEV-protease (pTEV) or lethal factor protease (pLF), plus 300ng empty pMSCV. (D) To assess IL-1 beta cleavage, cells were transfected as in C, but with 8ng of GFP-HA-NLRP1B and co-transfected with vectors encoding 200ng mCASP1 and 200ng mIL-1 beta . For panels B-D, data shown are representative of at least three similar experiments. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1006052), licensed under a CC-BY license. Not internally tested by R&D Systems.

Colocalization of phagosome markers with internalized Bb in WT and MyD88−/− BMDMs. a-b Confocal 40x images of WT (a) and MyD88−/− (b) BMDMs after 6 h stimulation with Bb at MOI 10:1, depicting colocalization of Bb-containing phagosomes with LAMP-1. White box indicates phagosome depicted in inset. Graph shows the intensity of each indicated pixel across the white line (distance on x-axis). Green is Bb, red is LAMP-1 and yellow is actin. c Quantitation of colocalization between Bb and LAMP-1 in 10 phagosomes of WT (black dots) and MyD88−/− (red dots) BMDMs by measuring intensity difference between LAMP-1 staining and Bb staining. d Western blot of protein lysate isolated from WT BMDMs after 6 h stimulation with Bb +/− ATP (C = cell lysate, S = supernatant) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/34000990), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>Differential effects of IL-1 beta neutralization on melanoma lung nodules in WT and DJ-1 KO mice.(A) IL-1 beta antibody (10μg) or control IgG was intravenously injected into mice once. The melanoma cells (6×104) were intravenously injected 30 minutes later. The mice were sacrificed three weeks later. Bar chart showed that the anti-IL-1 beta neutralizing antibody can effectively lower serum IL-1 beta levels as compared with control IgG in both WT and DJ-1 KO mice. Note that serum IL-1 beta levels were higher in DJ-1 KO mice than those in WT mice. (B) Inhibition of IL-1 beta neutralization on lung nodules formation in DJ-1 KO mice. Upper panels: melanoma nodules in lungs of WT (top) and KO (bottom) mice treated with control IgG (left) or neutralizing anti-IL-1 beta antibody (right). For each experimental condition, the gross and microscopic morphologies of tumor nodules are indicated by arrows, and are shown at left and right sites, respectively. Scale bars: 0.5 mm for lung photographs and 0.5 mm for H&E staining. Lower panel: Bar chart showed the summarized results of lung nodule numbers in WT and DJ-1 KO mice. Note that IL-1 beta neutralization enhanced and suppressed melanoma nodules formation in WT and DJ-1 KO mice, respectively. Data are presented as mean ± S.E.M. n = 5 for each group, * p < 0.05 compared with control IgG-treated WT mice, # p<0.05 compared with respective control IgG in WT and DJ-1 KO mice. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0115827), licensed under a CC-BY license. Not internally tested by R&D Systems.

Pro-IL-1 beta degradation is induced by the autophagy inhibitors chloroquine and bafilomycin A, and by the proteasome activator betulinic acid. (A) Chloroquine selectively attenuates IL-1 beta release from cardiac fibroblasts. The cells were incubated with LPS and/or 20 μM chloroquine phosphate as indicated for 18 h, then activated with 3 mM ATP for 60 min. IL-1 beta , TNF, MIP-2, and IL-6 were quantified in the conditioned media (n = 3). (B,C) Cardiac fibroblasts were incubated with LPS for 18 h with or without 20 μM chloroquine phosphate and pro-IL-1 beta quantified with western blot (n = 3). (D) Pro-IL-1 beta mRNA expression was quantified with qPCR (n = 3). (E,F) Cardiac fibroblasts were incubated with LPS for 20 h with or without 10% FCS or 20 μM chloroquine phosphate as indicated. The amount of total ubiquitinated protein was quantified with western blot (n = 4). (G,H) Cardiac fibroblasts were incubated with 10% FCS with or without 10 ng/mL LPS and/or 100 nM bafilomycin A1 for 20 h. Pro-IL-1 beta was quantified with western blot. (I,J) Cardiac fibroblasts were incubated with 10% FCS with or without 10 ng/mL LPS and/or 20 μM betulinic acid as indicated for 20 h. Pro-IL-1 beta was quantified with western blot. All columns are mean with SEM. *p < 0.05, **p < 0.01 (paired student t-test). Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31244838), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>The B6 variant of NLRP1B is not cleaved by LF but can form an inflammasome in response to proteolysis.(A) The amino acid sequence of the first 244 residues of NLRP1BB6 was aligned to the homologous sequence of NLRP1B129. The arrow above the alignment indicates the LF-cleavage site in NLRP1B129. Asterisks indicate sites of amino acid identity. (B) For detection of NLRP1B expression, 293T cells were transfected with the indicated amounts of and empty vector (V) or plasmids encoding GFP-HA-NLRP1B129 or GFP-HA-NLRP1BB6 (construct schematics and the predicted molecular weight of each protein is depicted in the upper panel). Cells were treated overnight with anthrax lethal toxin protein (LeTx, 1μg/ml) 24h post-transfection, and then lysates were analyzed by immunoblotting (IB) with the indicated antibodies. For NLRP1 expression and cleavage, CASP1 and IL1B were omitted to prevent cell death and resulting apparent differences of expression. For blots probed with anti-HA (NLRP1B), the lysates were not boiled prior to loading to prevent aggregation and smearing of full-length and FIIND-processed NLRP1B on the immunoblot. To visualize the N-terminal processed form of NLRP1B, the lysates were boiled and resolved on a separate gel. (C) 293T Cells were transfected with the same amount of titrated NLRP1B encoding plasmid and treated as in B, but transfections also included plasmids encoding mouse CASP1 (200ng), IL-1 beta (200ng) and 200ng of empty vector. (D) For detection of NLRP1B expression, 293T cells were transfected for 36h with 250ng of plasmids encoding WT 129 or B6 NLRP1B or mutants engineered to express the TEV-protease site. Each plasmid was co-transfected with either 100ng of empty vector (V) or plasmids encoding TEV-protease (pTEV) or lethal factor protease (pLF), supplemented with 300ng empty vector (pMSCV). Cells were not treated with LeTx and lysates were analyzed by immunoblotting as in (B). (E) Cells were treated as in C, but with 8ng of plasmids encoding NLRP1B, al

Rapamycin increases pro-IL-1 beta protein levels, rescues serum starvation-induced pro-IL-1 beta degradation, inhibits mTOR but induces no autophagy in LPS-stimulated cardiac fibroblasts. (A) IL-1 beta release was quantified in conditioned media from cardiac fibroblasts primed with 10 ng/mL LPS for 20 h with or without 500 nM rapamycin, as indicated, prior to activation with 3 mM ATP for 60 min. (B,C) Fibroblasts were primed with 10 ng/mL LPS for 20 h with or without 500 nM rapamycin and pro-IL-1 beta western blot performed (n = 3). (D,E) Control cells were incubated with 10% FCS, while serum deprived cells were incubated with or without 500 nM Rapamycin. Protein expressions were quantified with western blot (n = 4) (F,G). IL-1 beta and TNF-alpha were quantified in conditioned media from cardiac fibroblasts incubated with or without 10% FCS and 500 nM rapamycin and stimulated with LPS 10 ng/mL for 20 h prior to activation with 3 mM ATP for 60 min as indicated (n = 6) (H,I) IL-1 beta and TNF mRNA were quantified with PCR. (J–L) Cardiac fibroblasts were incubated with 10% FCS with or without 10 ng/mL LPS and/or 500 nM rapamycin for 20 h and mTOR (K) and p70 S6 kinase (L) activity were quantified with western blot (n = 5). (M,N) Cardiac fibroblasts primed with LPS (10 ng/mL) were incubated with 10% FCS (control) or no serum for 4 h. Cells were labeled with anti-LC3B and Hoechst and whole slides scanned with an automated immunofluorescence microscope. LC3B puncta in all cells in were automatically counted and ratio to number of Hoechst-labeled kernels calculated. Columns are mean with SEM of paired data normalized to control = 1 (n = 5). (O) Cardiac fibroblasts were incubated with 10% FCS, 10 ng/mL LPS, and/or 5 mM 3-methyladenine (3-MA) for 18 h and/or ATP 3 mM for 60 min as indicated (n = 3). Rapa: rapamycin. 3-MA: 3-methyladenine, ns: not significant, *p < 0.05, **p < 0.01, ****p < 0.0001 (paired student t-test). Image collected and cropped by CiteAb from the following publication (//pubm

SHP deficiency increases caspase-1 cleavage and IL-1 beta maturation.(a–e) LPS-primed BMDMs from SHP+/+ and SHP−/− mice were stimulated with ATP (5 mM, a,d), nigericin (15 μM, b,e), or transfected with polyinosine–polycytidylic acid (poly I:C, 5 μg ml−1, c) for the indicated durations. (a–c) Supernatants were collected and subjected to ELISA for interleukin (IL)-1 beta , IL-18 and tumour necrosis factor (TNF)-alpha . (d,e) IB analysis for IL-1 beta p17 or caspase-1 p10 in supernatants (SN), SHP, pro-IL-1 beta or pro-caspase-1 in whole-cell lysates (WCL). Actin was used as a loading control. *P<0.05; **P<0.01; ***P<0.001, compared with SHP+/+ cell cultures (two-tailed Student’s t-test). Data are the means±s.d. of values from four independent experiments (a–c). Data are representative of three independent experiments with similar results (d,e). Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms7115), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>ERS mediates M. bovis-induced inflammasome activation in vivo. (A) ELISA detection of IL-1 beta in serum samples from C57BL/6 mice treated with or without 4-PBA (18.6 mg/mouse/day) and infected with M. bovis (CFU 200) (n = 7). (B) Immunoblot analysis of NLRP3, AIM2, pro-IL-1 beta , and IL-1 beta in the lung tissues of M. bovis-infected mice in the presence or absence of 4-PBA. (C,D) Pathological lesions (H&E staining) in the lung of mice infected with M. bovis for 3 weeks (C) or 6 weeks (D) in the presence or absence of 4-PBA (18.6 mg/mouse/day) (n = 3). 4-PBA, 4-phenyl butyric acid, ERS inhibitor, 18.6 mg/mouse/day; CFU, colony forming units (n = 3). The results are shown as the mean ± SD. ***P < 0.001, n.s., not significant. P-values were analyzed by using Student's t-test (C,D). Scale bar = 100 μm. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30846986), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>POH1 negatively modifies K63-linked polyubiquitination of pro-IL-1 beta . a BMDMs stimulated with LPS for the indicated time periods were immunoprecipitated (IP) with an anti-POH1 or control IgG antibody and immunoblotted (IB) with the indicated antibodies. b HEK293T cells transfected with His-POH1 and either Flag-NLRP3, Flag-caspase-1 or Flag-ASC were subjected to IP with an anti-Flag antibody and IB with an anti-His antibody. c The lysates of HEK293T cells expressing Flag-POH1 and His-pro-IL-1 beta were IP with an anti-Flag antibody and IB with an anti-His antibody in cell lysates. d HEK293T cells transfected with Vsv-ASC, V5-tagged pro-caspase-1, HA-tagged K48-only or K63-only Ub and His-tagged pro-IL-1 beta , along with or without Flag-tagged POH1, were subjected to IP with an anti-V5 antibody or anti-His antibody and then IB with the indicated antibodies. ePoh1 delta /+ and Poh1 delta / delta BMDMs treated as indicated were subjected to IP with an anti-pro-IL-1 beta antibody and IB with the indicated antibodies. f HEK293T cells were transfected with Flag-caspase-1, Vsv-ASC, His-pro-IL-1 beta , Flag-POH1 (WT) or Flag-H113Q-POH1, along with HA-tagged K63-only Ub, then cells were subjected to IP with an anti-His or control IgG antibody and IB with the indicated antibodies. Similar results were obtained from three independent experiments Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30315153), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>BTK inhibitors and its dysfunctional mutation suppress NLRP3 inflammasome activation.(a) Enzyme-linked immunosorbent assay (ELISA) of human IL-1 beta in supernatants and immunoblot analysis of human IL-1 beta p17, caspase-1 p20/p22 in supernatants and pro-IL-1 beta in cell lysates of THP-1-Mφs that were pretreated with the indicated inhibitors for 30 min and then stimulated with alum for 6 h. ELISA of murine IL-1 beta (b,c) and TNF-alpha (b) in supernatants of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with alum for 3 h. (d,e) Immunoblot analysis of the indicated proteins (d) and ELISA of murine IL-1 beta and IL-6 in supernatants of LPS-primed peritoneal macrophages from Xid and WT mice stimulated with alum for 6 h. (f) ELISA of murine IL-1 beta in supernatants of LPS-primed murine peritoneal macrophages pretreated with LFM-A13 and/or Syk inhibitor (R406), then stimulated with alum for 3 h. (g) ELISA of murine IL-1 beta and immunoblot analysis of murine caspase-1 p20 in supernatant of LPS-primed murine peritoneal macrophages stimulated with the indicated NLRP3 inflammasome activators for 3 h. Immunoblot analysis of murine IL-1 beta p17, caspase-1 p20 in supernatants, pro-IL-1 beta , pro-caspase-1 and ASC in cell lysates (h), and ELISA of murine IL-1 beta in supernatants (i) of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with alum or poly(dA:dT) for 3 h. Data are representative of three independent experiments. Data are presented as mean±s.d. (triplicate). **P<0.01; ***P<0.003. Two-sided Student's t-test. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26059659), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>SHP inhibits the signal 2 activation of NLRP3 inflammasome.(a,b) LPS-primed BMDMs were treated with fenofibrate (10, 20, 50 μM for 4 h), or (c,d) LPS-primed THP-1 cells were treated with recombinant macrophage-stimulating protein (MSP 10, 50, 100 ng ml−1 for 4 h), and then activated with ATP (5 mM) for 30 min, followed by IB analysis of IL-1 beta p17 or caspase-1 p10 in supernatants (SN), pro-IL-1 beta or pro-caspase-1 in whole-cell lysates (WCL), with actin as a loading control. Data are representative of three independent experiments (a,c). Supernatants were collected and subjected to ELISA for IL-1 beta , IL-18, TNF-alpha and IL-8. Data are the means±s.d. of values from four independent experiments (b,d). *P<0.05; **P<0.01; ***P<0.001, compared with control (two-tailed Student’s t-test). FF, fenofibrate. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms7115), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>Effects of IL-1 beta neutralization on the accumulation of MDSCs in lungs.(A) Immunofluorescent photos showed that Gr-1+ (green)/CD11b+ (red) cells indicate MDSCs and increased in lungs of DJ-1 KO mice. Treatment with anti-IL-1 beta neutralizing antibody antagonized the accumulation of MDSCs. DAPI was used to stain nuclei. (B) Bar chart showed the summarized results of MDSCs in lungs of WT and DJ-1 KO mice. Note that the neutralizing antibody significantly reduced the number of MDSCs accumulated in lungs. n = 9 for each group, * p < 0.05 compared with control IgG-treated WT mice, # p<0.05 compared with respective control IgG in WT and DJ-1 KO mice. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0115827), licensed under a CC-BY license. Not internally tested by R&D Systems.

Pro-IL-1 beta maturation is regulated by POH1-mediated deubiquitination. a, b HEK293T cells were transfected with plasmids encoding Flag-caspase-1, Vsv-ASC, His-pro-IL-1 beta (WT) or His-pro-IL-1 beta -K133R with different amounts (125, 250, 500 ng) of plasmid encoding Flag-POH1 and 500 ng of control plasmid, the secretion of a IL-1 beta in the supernatants was quantified by ELISA 36 h after transfection; b the cell lysates were IB with the indicated antibodies. c Mutants of murine pro-IL-1 beta with replacement of various lysine residues. d-f HEK293T cells were transfected with Flag-POH1, V5-caspase-1, V5-ASC, His-pro-IL-1 beta (WT) or its mutants, along d with or e-f without HA-tagged K63-only Ub, then d cells were subjected to IP with an anti-His antibody and IB with the indicated antibodies. e The IL-1 beta levels in supernatants were measured by ELISA; or f the cell lysates were collected and IB with the indicated antibodies. g Proposed function of POH1 as a negative regulator in mediating IL-1 beta processing. POH1, upregulated by TLR3/4 activation, interacts with and deubiquitinates pro-IL-1 beta , resulting in restriction of IL-1 beta cleavage and secretion. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments. **p < 0.01, ***p < 0.001 (one-way ANOVA with Tukey’s post-hoc test) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30315153), licensed under a CC-BY license. Not internally tested by R&D Systems.

Serum starvation selectively induces pro-IL-1 beta degradation in cardiac fibroblasts. (A) Cells were incubated with 10 ng/mL LPS for 5 or 20 h with or without 10% FCS and western blot analysis of pro-IL-1 beta and NLRP3 performed. Blots are representative of 3 independent experiments. (B) Protein bands were quantified and the ratios to beta -actin normalized to mean control = 1 (n = 3). (C,D) Cardiac fibroblasts were incubated with or without 10% FCS and primed with LPS for 20 h. Western blot analysis of complex II (mitochondrial mass marker) and NLRP3- inflammasome protein components (NEK7, NLRP3, ASC, pro-caspase-1, and pro-IL-1 beta ) were performed. Bands were quantified and the ratios to beta -actin normalized to mean control = 1 (n = 4, for ASC n = 10). (E,F) Cardiac fibroblasts (120,000 cells per well seeded in 6 well plates) were incubated with 10 ng/mL LPS for 20 h, with or without 10% FCS, then stained with 500 nM MitoTracker Deep Red for 45 min before MitoTracker fluorescence intensity was quantified with flow cytometry analysis (633 nm laser). All cells were analyzed and all gated cells included in the analysis. The presented dot plots (E) show the gate in red and are representative for 9 biological repeats. Median MitoTracker intensity with and without 10% FCS are shown (F). All columns are mean with SEM. *p < 0.05,**p < 0.01 (paired student t-test). Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31244838), licensed under a CC-BY license. Not internally tested by R&D Systems.

Inflammasome assembly and mature IL-1 beta release in the spinal cord after sciatic nerve axotomy. a Speck-like costaining of NLRP3 and ASC in injured neurons 3 days after the injury (arrows). b Representative WB images of mature IL-1 beta and beta -actin proteins 3 days after unilateral axotomy of sciatic nerve. c Quantification of active IL-1 beta protein expression. N = 3 animals/group. The graph shows values normalized to beta -actin levels and relative to contralateral side. Mean values are shown on bars. Bars represent average ± SEM. **p < 0.01 (ANOVA with Bonferroni post hoc, ax. + vehicle vs. contralateral side). #p < 0.05, ##p < 0.01 (ANOVA with Bonferroni post hoc, compared to ax. + vehicle). Ax. axotomy Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35305649), licensed under a CC-BY license. Not internally tested by R&D Systems.

$GRA7-I activated inflammasomes in an ASC-binding dependent manner.(A) Bacterially purified 6xHis-GRA7-I and its mutants were analyzed by Coomassie blue staining (left) or IB with alpha His (right). (B) BMDMs from PKC alpha +/+ and PKC alpha -/- (left) or BMDMs were transduced with lentivirus-shRNA-NS or lentivirus-shRNA-ASC (MOI = 100) with polybrene (8 μg/mL) (right) for 2 days, the cells were stimulated with rGRA7 (5 μg/ml) and its mutants for the indicated times and culture supernatants were harvested and analyzed for cytokine ELISA for IL-1 beta and IL-18. Data shown are the means ± SD of five experiments. Significant differences (*P < 0.05; ***P < 0.001) compared with PKC alpha +/+ or shRNA-NS. (C) IB analysis for IL-1 beta p17, IL-18 p18, or caspase-1 p10 in supernatants (SN), ASC, NLRP3, pro-IL-1 beta , pro-IL-18, or pro-caspase-1 in whole-cell lysates (WCL). Actin was used as a loading control. (D) IB analysis of lysates of BMDMs as in B and C solubilized with Triton X-100–containing buffer, followed by cross-linkage of insoluble fractions with disuccinimidyl suberate to capture ASC oligomers and analysis of those fractions (I + DSS) and soluble fractions (S) with antibody to ASC. Actin was used as a loading control. (E) Fluorescence confocal images showing formation of speck-like ASC pyroptosomes in BMDMs from PKC alpha +/+ and PKC alpha -/- mice were stimulated with rGRA7 and its mutants for 18 h, fixed, immunostained with antibodies for ASC (Alexa 488). Scale bar, 10 μm. The data are representative of five independent experiments with similar results (A and C-E). Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1006126), licensed under a CC-BY license. Not internally tested by R&D Systems.$

<b>Biological Strategies Validation. </b>POH1 inhibits inflammasome-induced pro-IL-1 beta processing. a-c BMDMs from Poh1 delta /+ and Poh1 delta / delta mice were primed with LPS for 12–14 h and then stimulated with ATP (0.5 h), nigericin (Nig, 0.5 h), poly(dA:dT) (5 h) or flagellin (5 h) as indicated, a IL-1 beta , b IL-6 and c TNF alpha levels in supernatants were analysed by ELISA. d-f BMDMs lentivirally transduced with control (Vector) or POH1 were stimulated as in a, d IL-1 beta , e IL-6 and f TNF alpha levels in supernatants were analysed by ELISA. g, h BMDMs g derived from Poh1 delta /+ and Poh1 delta / delta mice or h infected by indicated lentiviruses were treated as in a, the cell lysates (CL) and supernatants (SN) were collected and immunoblotted with the indicated antibodies. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments. **p < 0.01, ***p < 0.001 (two-tailed Student’s t-test) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30315153), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>Quercetin inhibits NLRP3 and AIM2 Inflammasomes.LPS (500 ng/ml)-primed BMDM were treated with the indicated flavonoids and concentrations or vehicle (DMSO 0.01%), then stimulated with (A) 5 mM ATP (B) 10 μM Nigericin (C) 130 μg/ml Alum or (D) 400 ng/ml Poly (dA:dT) 30 minutes after treatment. (A–D) IL-1 beta and (E) TNF-alpha concentration in the culture supernatant were measured by ELISA. (B,F,G) LPS-primed BMDM were stimulated with Nigericin treated with quercetin 30 minutes before secondary stimulation. Supernatants and Lysate of BMDM were analyzed by immunoblotting. Cells were stained with FLICA and Caspase-1 activation was determined by FLICA positivity. Data shown are representative of two or more independent experiments (means ± SD) *p < 0.05, **p < 0.01, ***p < 0.001. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28148962), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>P. gingivalis and its OMVs differentially induce inflammasome signaling and pyroptosis in human macrophages. MDM were infected as before (2 h at MOI of 25:1, see Materials and Methods) with viable P. gingivalis (Pg), heat-killed-Pg (HK-Pg), OMVs, or heat-inactivated-OMVs (HI-OMVs) and (A) the activation of inflammasome components in the lysates was measured after 24 h by Western blot; beta -actin serves as a loading control throughout. Western blot data are representative of at least three independent experiments. (B) Production of IL-1 beta and IL-18 (by ELISA) from MDM was measured in the supernatant at 24 h. Data are mean ± SD from four independent experiments. Cell viability was determined by measuring extracellular LDH release and by 7-AAD exclusion (by flow-cytometry) at 24 h. 7-AAD is a membrane impermeant dye that is excluded from viable cells. The percent of 7-AAD positive (±SD) cells from three independent experiments was calculated relative to the untreated control. MDM treated with H2O2 (1 mM for 60 min) were included as positive control for 7-AAD. A representative histogram is shown. Cells were also treated where indicated with OMVs in the presence of KYT-1 and KYT-36 (10 μM of each). *p < 0.05: n.s, not significant. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28824884), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>P. gingivalis and its OMVs differentially induce inflammasome signaling and pyroptosis in murine macrophages. BMM were infected as before (2 h at MOI of 25:1, see Materials and Methods) with viable P. gingivalis (Pg), heat-killed-Pg (HK-Pg), OMVs, or heat-inactivated-OMVs (HI-OMVs) and (A) the activation of inflammasome components in the lysates [or supernatants (sup) where indicated] measured after 24 h by Western blot; beta -actin serves as a loading control throughout. Western blot data are representative of at least three independent experiments. (B) Production of IL-1 beta and IL-18 (by ELISA) from BMM was measured in the supernatant at 24 h. Data are mean ± SD from four independent experiments. Cell viability was determined by measuring extracellular LDH release and by 7-AAD exclusion (by flow-cytometry) at 24 h. 7-AAD is a membrane impermeant dye that is excluded from viable cells. The percent of 7-AAD positive (±SD) cells from three independent experiments was calculated relative to the untreated control. BMM treated with H2O2 (1 mM for 60 min) were included as positive control for 7-AAD. A representative histogram is shown. Cells were also treated where indicated with OMVs in the presence of KYT-1 and KYT-36 (10 μM of each). *p < 0.05. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28824884), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Genetic Strategies Validation. </b>Caspase-1 but not gasdermin-D processing depends on ASC oligomerization.(a) Western blot analysis for cleaved caspase-1 p20, IL-1 beta p17, and HMGB-1 in cell supernatants (SN) and pro-caspase-1, pro-IL-1 beta and HMGB-1 in cell lysates (lys) of LPS-primed immortalized Asc−/− BMDMs or Asc−/− BMDMs expressing ASCFL, ASCY59A or ASCE80R 3 h after poly(dA:dT) transfection (1 μg ml−1). (b) Release of LDH from LPS-primed immortalized Asc−/− BMDMs or Asc−/− BMDMs expressing ASCFL, ASCY59A or ASCE80R, or derived Casp1 knockouts 3 h after poly(dA:dT) transfection (1 μg ml−1). (c) Western blot analysis for processing of full-length gasdermin-D (GSDMDFL) into the active N-terminal fragment (GSDMDN-term) in combined lysates and supernatants (lys + SN) of LPS-primed immortalized Asc−/− BMDMs expressing ASCFL, ASCY59A or ASCE80R, or derived Casp1 knockouts 3 h after poly(dA:dT) transfection (1 μg ml−1). Arrowhead, gasdermin-DNterm p30; *a cross-reacting band. (d) Release of LDH from LPS-primed primary C57BL/6 WT (WT), Casp1−/−/Casp11−/− or Asc−/− BMDMs infected with S. Typhimurium (multiplicity of infection (MOI)=10, 1 h). (e) Western blot analysis for processing of full-length gasdermin-D (GSDMDFL) into the active N-terminal fragment (GSDMDN-term) in combined lysates and supernatants (lys+SN) of LPS-primed primary C57BL/6 WT (WT), Casp1−/−/Casp11−/− or Asc−/− BMDMs infected with S. Typhimurium (MOI=10, 1 h) or left uninfected. Arrowhead, gasdermin-DNterm p30; *a cross-reacting band; ** a S. Typhimurium-specific cross-reactive band. See also Supplementary Figs 8 and 9. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms11929), licensed under a CC-BY license. Not internally tested by R&D Systems.

Nimbidiol treatment mitigated elevated pro-inflammatory cytokine and chemokine in the diabetic kidney. Representative images from (A) Semi-quantitative RT-PCR analyses showing gene expression and (B) western blot analyses showing protein expression of TNF-alpha , IL-1 beta and MCP-1 in the kidney. The bar diagrams represent the fold change from WT + Saline. Data are mean ± SD (n = 6/group). *p < 0.05 versus WT + Saline, WT + Nimbidiol and Akita + Nimbidiol, †p < 0.05 versus Akita + Saline. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/36522378), licensed under a CC-BY license. Not internally tested by R&D Systems.

Loss IL-1 beta K133 ubiquitylation or the IL-1 beta K133:D129 salt bridge stabilizes IL-1 beta .a WT Il1b, Il1bK133A and Il1bD129A cDNAs were expressed in 293T cells and 48 h after transfection cells exposed to cycloheximide (CHX, 20 μg/ml) for up to 7 h. Cell lysates were immunoblotted for IL-1 beta levels. The arrow indicates pro-IL-1 beta . Actin is included as a loading control. One of three experiments. b WT Il1b cDNA was transfected into 293T cells and after 48 h cells were cultured with MG132 (20 μM) as indicated for 15 min prior to cycloheximide (CHX, 20 μg/ml) treatment for up to 7 h. Immunoblots were performed on total cell lysates for IL-1 beta protein levels. The arrow indicates pro-IL-1 beta . Actin is included as a loading control. One of two experiments. c 293T cells expressing WT IL-1 beta , IL-1 beta K133R, IL-1 beta K133A, IL-1 beta D129A and IL-1 beta D117A were co-transfected with Caspase-1 (250 or 500 ng) in the presence or absence of the pan-caspase inhibitor Z-VAD-fmk (50 μM) for 24 h. Immunoblots were performed on cell supernatants and total cell lysates for the indicated proteins. One of 2–3 experiments. d, e 293T cells expressing WT IL-1 beta , IL-1 beta K133R, IL-1 beta K133A, IL-1 beta S134A (phospho-ablating) and IL-1 beta S134E (phospho-mimetic) were co-transfected with 200 and 400 ng of d Caspase-1 or e Caspase-8, as indicated, for 24 h. IL-1 beta secretion was analyzed by ELISA. Data are the mean + S.D of three independent experiments. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/33976225), licensed under a CC-BY license. Not internally tested by R&D Systems.

Identification of new NLRP3 inflammasome activators. a Schematic of activator-suppressor screens for IL-1 beta release. b Effect of the top 3 activators (6 h treatment, this notation is used throughout the figure legends) on IL-1 beta release (nigericin; positive control). c Immunoblot of biologically active IL-1 beta (p17) and caspase-1 (Casp1; p20) following activator treatment (3 h). Data represent two independent experiments; full gel images are in Supplementary Figure 14. d Effect of gene knockout of NLRP3 inflammasome components on IL-1 beta release (6 h). Guide RNA corresponding to non-targeting negative controls (N.Ctl) or gene-specific targets are indicated. e Effect of activators on LDH release (1 h, 3 h, 6 h). f Effect of P2RX7 antagonist on IL-1 beta release (1 h A438079 pretreatment followed by 6 h activator treatment in the presence of A438079). Data are mean ± s.d.; n = 3 (b, f), 6 (e), or 8 (d) biological replicates from one independent experiment; data are representative of two independent experiments. P values were determined by two-way ANOVA followed by Tukey’s multiple testing (d, e). *P < 0.0001 compared to N.Ctl1 (d) or no activator control of each time point (e). THP-1 macrophages (b, c, e, f) or THP-1 macrophages expressing guide RNA (d) were used for measurements. Activators were used at 1 μM (niclosamide), 2.5 μM (AG-879), 1 μM (ouabain), 20 μM (nigericin), or 5 mM (ATP) (c–f) Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/30740538), licensed under a CC-BY license. Not internally tested by R&D Systems.

Niclosamide-biased suppressors prevent intracellular potassium loss. a Results of suppressor screens using nigericin (20 μM) or niclosamide (5 μM) as activators. Yellow circles indicate the top 60 validated activators. b, c Effect of niclosamide-biased suppressors on IL-1 beta release. d Effect of suppressors on LDH release. e Immunoblot of biologically active IL-1 beta (p17) and caspase-1 (Casp1; p20) after suppressor treatment. Data represent two independent experiments. Full gel images are shown in Supplementary Figure 14. f FACS analysis of mitochondrial ROS. Data represent three independent experiments. g Effect of suppressors on intracellular potassium level. h Rescue of IL-1 beta release by nigericin following suppressor treatment. Numbers (1–3) represent experimental scheme illustrated above the figure panel. Data are mean ± s.d.; n = 3 (b, c), 4 (g), 5 (h), or 6 (d) biological replicates from one independent experiment; data are representative of two independent experiments. P values were determined by two-way ANOVA followed by Tukey’s multiple testing. *P < 0.05, ***P < 0.001, ****P < 0.0001 as indicated. THP-1 macrophages (a–e, h) or THP-1 macrophages expressing NLRP3 guide RNA (f, g) were used for measurements. 1 h suppressor treatment (10 μM DPI, 10 μM LP44, or as indicated) was followed by 1 h (f) or 6 h (b–e, g–h) activator treatment (1 μM niclosamide, 20 μM nigericin, 1 μM ouabain, or 250 μg mL−1 silica) in the presence of suppressors (b–h). Nigericin rescue (h) (20 μM) for 1 h was performed following 6 h niclosamide treatment Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/30740538), licensed under a CC-BY license. Not internally tested by R&D Systems.

Cleavage of NLRP1B is sufficient to promote inflammasome activation.A) 293T cells were transfected with WT, TEV-site2 or TEV-site1 GFP-HA-NLRP1B along with empty vector, TEV expression vector, or a LF expression plasmids. In all conditions cells were also co-transfected with Casp1 and Il1b expression vectors. Cleavage of GFP-HA-NLRP1B and IL-1 beta was determined 24 h post transfection. B) Immortalized B6 macrophages were transduced with a retrovirus encoding the indicated GFP-HA-NLRP1B form followed by a sequential transduction with a TEV-expression retrovirus co-expressing THY1.1. Percent transduction was determined by measuring expression of the respective retroviral integration markers (GFP and anti-THY1.1-PE-Cy7) by flow cytometry, and are expressed in relative fluorescent units (RFU). The numbers within each quadrant represent the percentage of live cells within the respective quadrant. C) RAW264.7 macrophages were transduced with GFP-HA-NLRP1B and a Tet-On construct expressing the indicated gene. Cells were treated with 5 µg/ml doxycycline for 20 h and supernatants were assayed for LDH release. D) 293T cells were transfected with empty vector, FL-NLRP1B-HA, the truncated NLRP1B-HA, or delta LRR HA-NLRP1B, along with Casp1 and Il1b and assayed by immunoblotting. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/23818853), licensed under a CC-BY license. Not internally tested by R&D Systems.

LPS-induced IL-1 beta is increased in Il1bK133R/K133R mice in vivo.a BMDMs derived from mice of the indicated genotypes were treated with LPS (100 ng/ml) for 3 h, followed by proteasome inhibition (PS341, 10 μM) for an additional 3 h. Biotin conjugated IL-1 beta antibody was used to purify IL-1 beta from cell lysates and its modification analyzed using the indicated ubiquitin (Ub) chain-specific antibodies. Cell lysates from the same experiment were run in triplicate with roman numerals on the bottom right of the blots indicating the membrane probed. One of two independent experiments. b BMDMs were primed with LPS (100 ng/ml) for 3 h followed by treatment for a further 3 h with PS341 (10 μM). Cells were subsequently lysed in buffer containing either PS341 (10 μM), or PS341 and PR619 (10 μM), as indicated. Recombinant caspase-1 (5 Units) was added and cell lysates incubated at 37 °C for 80 min. Subsequently, ubiquitylated proteins were purified by Tandem Ubiquitin Binding Entities (TUBEs) and analyzed by immunoblot. One of two independent experiments. c–f WT and Il1bK133R/K133R mice were injected intra-peritoneally with 100 μg of LPS. After 2 h serum and peritoneal fluid was harvested and analyzed by ELISA for levels of c IL-1 beta , d TNF, and e IL-6. Data are mean ± SEM, symbols represent individual mice, n ≥ 19 mice/genotype from three pooled independent experiments for c, d and n ≥ 14 for e from 2 to 3 pooled experiments. P-values were calculated using an unpaired two-tailed t-test for comparisons between genotypes. f Peritoneal fluid was analyzed by immunoblot for the indicated proteins. Each lane represents an individual mouse. n = 5–6 mice per genotype. One of two independent experiments (analysis of nine additional WT and 19 Il1bK133R/K133R mice are shown in Supplementary Fig. 7b). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/33976225), licensed under a CC-BY license. Not internally tested by R&D Systems.

PEG-Arg1 treatment reduced retinal inflammation and reversed MΦ/microglia soma enlargement. (A,C) Representative Western blots of the pro-inflammatory cytokines IL-1 beta and TNF- alpha in retinal protein lysates from PEG-A1-treated db/db mice with quantification (B,D, respectively) compared with the PEG-treated db/db controls (n = 6/group, * p < 0.05, ** p < 0.01). (E) Retinal flat-mounts imaged at the level of the superficial vascular plexus labeled with isolectin B4 (vasculature) and Iba-1 (MΦ/microglia) with Iba-1 in the enlarged inset (boxed in white) showing exceptionally enlarged perivascular MΦ/microglia. (F) Quantification of MΦ/microglia soma size, demonstrating significant size reductions in the db/db mice treated with PEG-Arg1 compared with the vehicle-treated controls (n = 3 mice/group, * p < 0.05). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/36139465), licensed under a CC-BY license. Not internally tested by R&D Systems.

ATP and nigericin can augment proteasomal-mediated IL-1 beta degradation.a BMDMs were primed with Lipopolysaccharide (LPS, 50 ng/ml) for 3 h and stimulated with ATP (5 mM) and Nigericin (10 μM) for up to 2 h. Total cell lysates were analyzed by immunoblot. One of three experiments. b, cCaspase-1−/− BMDMs were primed with LPS (50 ng/ml) for 3 h, and in the last 30 min of priming treated with MG132 (20 μM), as indicated. Cells were treated with b ATP (5 mM) and c Nigericin (10 μM) for up to 90 min. Immunoblots were performed on total cell lysates to detect the specified proteins. One of three experiments. d WT, Nlrp3−/−, Asc−/− and Caspase-1−/− BMDMs were primed with LPS (50 ng/ml) for 3 h and stimulated with ATP for 20 min. Ubiquitylated (Ub.) proteins were purified from cell lysates by Tandem Ubiquitin Binding Entities (TUBEs). Immunoblots were performed on cell lysates and TUBE isolated ubiquitylated proteins for the indicated proteins. One of two experiments. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/33976225), licensed under a CC-BY license. Not internally tested by R&D Systems.

Loss of K133 ubiquitylation in mice stabilizes IL-1 beta and increases IL-1 beta activation.a. WT and Il1bK133R/K133R BMDMs were primed with Lipopolysaccharide (LPS, 50 ng/ml) or Pam3Cys-Ser-(Lys)4 (Pam3Cys, 500 ng/ml) for 3 h with Q-VD-OPh (20 μM) added in the last 30 min of priming. Cells were then treated with the protein synthesis inhibitor cycloheximide (CHX, 20 μg/ml) for up to 6 h. Total cell lysates were analyzed for the indicated proteins by immunoblot. One of four experiments. b, c WT and Il1bK133R/K133R BMDMs were primed with LPS (50 ng/ml) for 3 h and then treated with ATP (5 mM) and Nigericin (10 μM) for the times indicated in Opti-MEM media. b IL-1 beta and c TNF levels were measured in cell supernatants by ELISA. Data are the mean + SD of two independent experiments combined using three mice of each genotype. d WT and Il1bK133R/K133R BMDMs were primed with LPS (50 ng/ml) for 3 h and treated with ATP (5 mM) and Nigericin (10 μM) for up to 60 min in Opti-MEM media. Cell supernatants and lysates were analyzed by immunoblot for the indicated proteins. One of three experiments. e WT and Il1bK133R/K133R neutrophils sorted from bone marrow were primed with IFN gamma (50 ng/ml, 1 h) and then treated with LPS (100 ng/ml) for a further 3 h. Cells were then stimulated with Nigericin (10 μM) for up to 6 h, as indicated. Cell supernatants and lysates were analyzed by immunoblot for the specified proteins. One of three experiments. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/33976225), licensed under a CC-BY license. Not internally tested by R&D Systems.

Co-localization of inflammasome components&production of active interleukin-1 beta (IL-1 beta )&IL-18 (IL-18) in the XII. nucleus. (D) Representative WB images of IL-1 beta &IL-18 proteins in of the hypoglossal nuclei after unilateral axotomy of corresponding brain nerve. Arrows indicate pro-forms, arrowheads show active cytokines. Image collected & cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/33328988), licensed under a CC-BY license. Not internally tested by R&D Systems.

EhaF inhibits innate immune responses to EHEC infection.a–c, e–l Secretion of indicated cytokines by C57BL/6 or Tlr4−/− BMDMs infected with EHEC, the indicated isogenic mutant, or complement strains at an MOI of 50 (MOI of 50 for all infections hereafter unless otherwise indicated) or treated with 0.5 μg/ml LPS or 0.5 μg/ml Pam3CSK4 for 6 h (b, c, f, g, h, j, k) or 18 h (a, e, i, l). d Immunoblot for pro IL-1 beta and beta -actin in the lysates of BMDMs infected with EHEC or delta OI-14-15 for the indicated times. m, n Fold increase in the expression of indicated genes in C57BL/6 or Tlr4−/− BMDMs infected with the indicated E. coli strains or treated with 0.5 μg/ml Pam3CSK4 relative to uninfected BMDMs (medium) as determined by real time quantitative PCR at 2 h post-stimulation. o, p Secretion of indicated cytokines by Caco2 cells infected with the indicated (on x-axis) MOI of EHEC or delta EhaF for 24 h. a–c, e–p, Data (mean ± SEM) were from three independent experiments and each dot is a mean of each experiment’s technical replicates. d Immunoblot from one experiment representative of three independent experiments is shown. Statistical significance was assessed using one-way ANOVA (a–c, e–i) or two-way ANOVA (j–p) followed by Tukey’s post-test. p < 0.05 indicated statistical significance. Multiplicity adjusted p values are presented. Source data are provided as a Source Data file. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37041208), licensed under a CC-BY license. Not internally tested by R&D Systems.

Elevated retinal inflammation and MΦ/microglia soma enlargement in db/db mice. (A) Representative Western blots of IL-1 beta in retinal protein lysates with quantification in (B), demonstrating significant increases in IL-1 beta (n = 10) expression in db/db mice compared with the lean DB/+ controls. (C) Retinal flat-mounts imaged at the level of the superficial vascular plexus and labeled with isolectin B4 (vasculature) and Iba-1 (MΦ/microglia). White arrows point to the perivascular macrophages with Iba-1 in the enlarged inset (boxed in white) showing exceptionally enlarged perivascular MΦ/microglia. (D) Quantification of MΦ/microglia soma size, demonstrating a significant size increase in db/db mouse retinas compared with the lean controls (n = 3 mice/genotype, * p < 0.05). Data are presented as the mean ± SEM. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/36139465), licensed under a CC-BY license. Not internally tested by R&D Systems.

Rac1 haploinsufficiency rescues arthritis & inflammatory signaling in Pggt1b delta / delta mice. d WBs showing levels of mature Il-1 beta & caspase-1 in supernatants (Sup), & pro-Il-1 beta & pro-caspase-1 in lysates (Lys) of LPS (200 ng/ml) stimulated BM macrophages; tubulin in lysates was used as a loading control. The antibiotic nigericin (28 mM) was used as a positive control for inflammasome-mediated caspase-1 activation & Il-1 beta production. Image collected & cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/31484924), licensed under a CC-BY license. Not internally tested by R&D Systems.

Examining the role of microglia-derived IL-1 beta in promoting astrocyte TIMP-1 expression. A. Determination of IL-1 beta in LPS-activated MG-CM. Microglial cultures were treated with LPS for 2 days, then subject to either six rounds of IL-1 beta immunodepletion, or mock depletion, as described in Materials and Methods, and samples analyzed for the presence of IL-1 beta . Note that IL-1 beta was detected in the LPS-activated MG-CM before, but not after the IL-1 beta immunodepletion, demonstrating the effectiveness of the immunodepletion strategy. B and C. The effect of removing IL-1 beta from LPS-activated MG-CM on astrocyte TIMP-1 production. Pure astrocyte cultures received either: LPS, microglia + LPS, LPS-activated MG-CM (mock depleted) or LPS-activated MG-CM depleted of IL-1 beta . Note that the presence of LPS-activated microglia or LPS-activated MG-CM strongly induced TIMP-1 expression in astrocytes, but this effect was markedly attenuated after IL-1 beta removal from the MG-CM. * p < 0.02. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/21631912), licensed under a CC-BY license. Not internally tested by R&D Systems.

AGER is required for caspase-11 inflammasome activation. (A–C) Analysis of cytotoxicity (A), IL-1 beta release (B), and IL-18 release (C) in LPS-primed BMDMs after HHD treatment (500 ng/ml, 16 h) in the absence or presence of FPS-ZM1 (100 nM, 500 nM, and 1 μM). n = 3, data expressed as means ± SD of three independent experiments, *P < 0.05 vs. HHD group, t test. (D–F) Analysis of cytotoxicity (D), IL-1 beta release (E), and IL-18 release (F) in indicated LPS-primed BMDMs after HHD treatment (500 ng/ml, 16 h), LPS electroporation (LPSe; 1 μg, 16 h), or E. coli infection (MOI = 25, 16 h). n = 3, data expressed as means ± SD of three independent experiments, *P < 0.05 vs. WT group, t test. (G) Western blot analysis of indicated proteins in the supernatant (SN) or cell lysate in LPS-primed BMDMs after HHD treatment (500 ng/ml, 16 h) or LPS electroporation (LPSe; 1 μg, 16 h). Western blot data represent two independent experiments. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/31440260), licensed under a CC-BY license. Not internally tested by R&D Systems.

GSDMD is required for nuclear DAMP complex-induced pyroptosis. (A) Western blot analysis of indicated proteins in the supernatant (SN) or cell lysate in indicated LPS-primed BMDMs after HHD treatment (500 ng/mL, 16 h). (B–G) Analysis of cytotoxicity (B), IL-1 beta (C), IL-18 (D), TNF (E), IL-6 (F), and IL-12 (G) release in indicated LPS-primed BMDMs after HHD treatment (500 ng/mL, 16 h) in the absence or presence of the overexpression of GSDMD WT or D275A cDNA. n = 3, data expressed as means ± SD of three independent experiments, *P < 0.05, t test. Western blot data represent two independent experiments. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/31440260), licensed under a CC-BY license. Not internally tested by R&D Systems.

Charged liposomes activate the NLRP3 inflammasome to induce IL-1 beta secretion ELISA for IL-1 beta from the supernatants of (a) LPS-primed wild-type BMDMs or (b) PMA-primed THP1 cells that were stimulated with either the indicated liposomes (30 μg/ml for BMDMs and 50 μg/ml for THP1 cells) or alum (250 μg/ml for BMDMs and 500 μg/ml for THP1 cells). (c) Immunoblots of procaspase-1, activated caspase-1 (p10), pro-IL-1 beta and cleaved IL-1 beta (p17) in the culture supernatants (Sup) and cell lysates (Lys) from LPS-primed BMDMs after stimulation with indicated liposomes or alum. (d) IL-1 beta from supernatants of LPS-primed BMDMs that were pretreated with caspase-1 inhibitor (z-YVAD-fmk, 10 μM) for 45 min followed by stimulations with indicated liposomes or alum. (e) ELISA for IL-1 beta from the supernatants of LPS-primed or unprimed wild-type BMDMs that were stimulated with indicated liposomes (30 μg/ml) or alum (250 μg/ml). (f) The IL-1 beta levels from the supernatants of LPS-primed immortalized mouse macrophages from wild-type, Nlrp3−/−, Asc−/−, Caspase-1−/− or Nlrc4−/− mice that were stimulated with liposomes (70 μg/ml). (g) ELISA for IL-1 beta from the supernatants of LPS-primed wild-type BMDMs that were cultured in 150 mM of KCl or NaCl followed by stimulation with liposomes or alum. Data in a, b and d–g are shown as mean ± s.d., and all data are representative of at least three independent experiments. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/23511475), licensed under a CC-BY license. Not internally tested by R&D Systems.

AGER-mediated lipid peroxidation promotes caspase-11 inflammasome activation. (A–C) Analysis of lipoxygenase activity (A), MDA level (B), and 4-HNE level (C) in indicated LPS-primed BMDMs after HHD treatment (500 ng/ml, 16 h) or LPS electroporation (LPSe; 1 μg, 16 h) in the absence or presence of FPS-ZM1 (1 μM). n = 3, data expressed as means ± SD of three independent experiments, *P < 0.05, t test. (D–F) Analysis of cytotoxicity (D), IL-1 beta release (E), and IL-18 release (F) in indicated LPS-primed BMDMs after HHD treatment (500 ng/ml, 16 h) or LPS electroporation (LPSe; 1 μg, 16 h) in the absence or presence of zileuton (5 μM). n = 3, data expressed as means ± SD of three independent experiments, *P < 0.05, t test. (G) Western blot analysis of indicated proteins in the supernatant (SN) or cell lysate in LPS-primed BMDMs after HHD treatment (500 ng/ml, 16 h) or LPS electroporation (LPSe; 1 μg, 16 h). Western blot data represent two independent experiments. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/31440260), licensed under a CC-BY license. Not internally tested by R&D Systems.

IL-1 beta promotes priming and activation of the NLRP3 inflammasome in HUVEC. The expression of the NLRP3 inflammasome components (A) NLRP3 and (B) pro-IL-1 beta was determined by Western blot in total lysates from HUVEC stimulated for 18-24 h with IL-1 beta . Representative blots are shown on top of the corresponding graphs, where beta -Actin was employed as loading control (n= 4-5; *p<0.05 vs. untreated cells by unpaired t-test). (C) The NLRP3 inflammasome activation was determined by the observation of ASC specks by indirect immunofluorescence after treatment with IL-1 beta alone or in presence of MCC 950 (1 µM). The percentage of ASC specks per cell culture field was quantified under a fluorescence microscope (n= 3-4; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1 beta -treated cells, by two-way ANOVA and Tukey post-hoc test). (D) Confocal representative photographs (630X magnification) where white arrowheads show the location of toroidal-shaped ASC specks as detected by a specific antibody against ASC (red), while cell nuclei were counterstained with DAPI (blue). (E) IL-1 beta , the final product of the NLRP3 inflammasome activation was measured in the supernatant of treated cells by ELISA (n= 4-5; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1 beta -treated cultures levels by two-way ANOVA and Tukey post-hoc test). HUVEC were treated with 2.5 ng/ml IL-1 beta in all experiments. All data are shown as mean ± SEM and expressed as percentage of IL-1 beta -induced levels. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35111374), licensed under a CC-BY license. Not internally tested by R&D Systems.

Manoalide inhibits the NLRP3 inflammasome activation. (A-C) BMDMs were primed with LPS for 3 h and then were pretreated with 125, 250, 500 nM manoalide before stimulation with nigericin for 15 min (A) ELISA of mature IL-1 beta in supernatant from BMDMs. (B) Western blots analysis of supernatant (SN) and cell lysates (Input) from BMDMs. (C) LDH release in the supernatant from BMDMs. (D) ELISA of mature IL-1 beta and (E) Western blots of SN and Input from BMDMs primed with LPS and stimulated with nigericin, MSU and ATP with or without 500 nM manoalide. (F-H) BMDMs were primed with Pam3K for 3 h and then were pretreated with 125, 250, 500 nM manoalide before stimulation with cytosolic LPS (cLPS) for 16 h. (F) Production of mature IL-1 beta in supernatant from BMDMs. (G) Western blots analysis of supernatant (SN) and cell lysates (Input) from BMDMs. (H) LDH release in the supernatant from BMDMs. (I) ELISA of mature human IL-1 beta in supernatant from PBMCs primed with LPS for 3 h and then were pretreated with 125, 250, 500 nM manoalide before stimulation with nigericin. Data are derived from three independent experiments (A,C,D,F,H,I) and displayed as mean and SEM (n = 6) or represent three independent experiments (B,E,G). Statistically significant difference was calculated by unpaired Student’s t-test: ***p < 0.001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35252186), licensed under a CC-BY license. Not internally tested by R&D Systems.

The activation of NLRP3 by IL-1 beta is mediated by NF-kappa B. The expression of (A) phosphorylated p65 (p-p65) and total p65 (p65), used as a marker of NF-kappa B activation, and the NLRP3 inflammasome components (B) NLRP3, (C) pro-IL-1 beta and (D) mature IL-1 beta levels was determined by Western blot in total lysates from HUVEC stimulated for 18 h with IL-1 beta in presence or absence of PDTC (100 µM). Representative blots are shown on top of the corresponding graphs, where beta -Actin was employed as loading control (n= 5-7; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1 beta -treated cells, by two-way ANOVA and Tukey post-hoc test). All data are shown as mean ± SEM and expressed as percentage of IL-1 beta -induced levels. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35111374), licensed under a CC-BY license. Not internally tested by R&D Systems.

PL dose-dependently inhibits nigericin-induced NLRP3 inflammasome activation. (A) Cell viability of PL (1–40 μM) in peritoneal macrophages. (B–D) ELISA of IL-1 beta (B), TNF-alpha (C), and release of LDH (D) in supernatants from LPS-primed mouse peritoneal macrophages treated with 1–10 μM PL and stimulated with nigericin. (E) Immunoblot of supernatants or cell lysates from LPS-primed mouse peritoneal macrophages treated with 1–10 μM PL and stimulated with nigericin. All data were representative of three independent experiments. Values shown are mean ± SD. For statistical analysis, A–D were analyzed using one-way ANOVA and the Bonferroni test. *p < 0.05; **p < 0.01; ***p < 0.001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35095532), licensed under a CC-BY license. Not internally tested by R&D Systems.

Manoalide has no effects on the activation of AIM2 inflammasome, IPAF inflammasome or Pyrin inflammasome. (A) ELISA of mature IL-1 beta and (B) Western blots of SN and Input from BMDMs primed with LPS and stimulated with nigericin, Poly (A:T) or salmonella (MOI = 10) with or without 500 nM manoalide. (C) ELISA of mature IL-1 beta in SN from BMDMs primed with LPS and then were pretreated with 500 nM manoalide before stimulation with C3 toxin. Data are derived from three independent experiments (A,C) and displayed as mean and SEM (n = 6) or represent three independent experiments (B). Statistically significant difference was calculated by unpaired Student’s t-test: ***p < 0.001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35252186), licensed under a CC-BY license. Not internally tested by R&D Systems.

149-01 does not affect the activation of AIM2, NLRC4, or Pyrin inflammasomes. (A–C) BMDMs were first primed with LPS, then treated with or without 149-01 (600 nM) for 30 min, lastly stimulated with nigericin, poly (A/T), Salmonella or C3 toxin. (A,C) IL-1 beta releases in supernatants were detected by ELISA. (B) Active IL-1 beta and p20 in supernatants and pro-IL-1 beta , pro-caspase-1 and beta -actin in cell lysates were measured by western blot. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m (n = 6) (A,C), or are representative of three independent experiments (B). One-way ANOVA was applied to calculate statistical significance: ***p <.001, NS, not significant. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35250572), licensed under a CC-BY license. Not internally tested by R&D Systems.

Identification of 149-01 as a highly potent inhibitor for NLRP3 inflammasome. (A) 149-01 structure. (B–F) BMDMs were first primed with LPS for 3 h, then pretreated with indicated doses of 149-01 (200-600 nM) for 30 min, lastly stimulated with nigericin. (B) IL-1 beta releases in supernatants were detected by ELISA. (C) Active IL-1 beta and p20 (cleaved caspase-1) in supernatants (SN) and pro-IL-1 beta , pro-caspase-1 and beta -actin in cell lysates (Input) were measured by western blot. (D) The release of LDH in supernatants. (E) IL-6 and (F) TNF-alpha secretion levels in supernatants were determined by ELISA. (G,H) BMDMs were first primed with LPS, then treated with or without 149-01 (600 nM) for 30 min, lastly stimulated with nigericin, ATP or MSU. (G) IL-1 beta releases in supernatants were detected by ELISA. (H) Active IL-1 beta and p20 in supernatants and pro-IL-1 beta , pro-caspase-1 and beta -actin in cell lysates were measured by western blot. (I,J) PBMCs were first primed with LPS for 3 h, then pretreated with indicated doses of 149-01 for 30 min, lastly stimulated with nigericin. (I) IL-1 beta and (J) TNF-alpha secretion levels in supernatants were determined by ELISA. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m (n = 6) (B,D–G,I,J), or are representative of three independent experiments (C,H). One-way ANOVA was applied to calculate statistical significance: ***p <.001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35250572), licensed under a CC-BY license. Not internally tested by R&D Systems.

FA inhibits I/R-induced myocardial injury by inhibiting NLRP3 inflammasome activation. (A–F) Expression of NLRP3, pro-caspase-1, caspase-1, pro-IL-1 beta , and IL-1 beta in the heart tissue of WT mice was detected by western blotting. (G) HE staining of heart tissue sections in WT mice and Nlrp3−/−mice. (H–K) Expression of IL-1 beta , caspase-1, and NLRP3 in heart tissues of WT mice and Nlrp3−/− mice in each experimental group, detected by western blotting. (L–O) Expression of CK-MB, LDH, troponin-1, and NT-proBNP in the serum of each experimental group in WT mice and Nlrp3−/−mice, determined by ELISA. Data are expressed as the means ± sd from three independent experiments or are representative of three independent experiments. **p < 0.01, ***p < 0.001; ns indicates no significance. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35496295), licensed under a CC-BY license. Not internally tested by R&D Systems.

PL inhibits ATP or MSU-induced NLRP3 inflammasome activation. (A–C) ELISA of IL-1 beta (A), TNF-alpha (B), and release of LDH (C) in supernatants from LPS-primed mouse peritoneal macrophages treated with 10 μM PL or DMSO and stimulated with ATP or MSU. (D) Immunoblot of supernatants or cell lysates from LPS-primed mouse peritoneal macrophages treated with 10 μM PL and stimulated with indicated stimuli. All data were representative of three independent experiments. Values shown are mean ± SD. For statistical analysis, A, B, and C were analyzed using two-way ANOVA and the Bonferroni test; **p < 0.01; ***p < 0.001; ****p < 0.0001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35095532), licensed under a CC-BY license. Not internally tested by R&D Systems.

The inflammasome NLRP3 is implicated in the pro-senescence effect of IL-1 beta in endothelial human cells. HUVEC were treated with IL-1 beta (2.5 ng/ml) alone or in presence of the NLRP3 inflammasome inhibitor MCC 950 (1 μM) for 18-24 h. (A) SA-beta -Gal positive stained cells were quantified by manual scoring by a blind observer after the indicated treatments. (B) Representative phase-contrast images are shown SA-beta -Gal positive cells stained in blue (black arrowheads). Data are expressed as percentage of the number of senescent cells stained in non-treated cells (n= 5-7; *p<0.05 vs. untreated cells; #p<0.05 vs IL-1 beta -treated cultures levels by two-way ANOVA and Tukey post-hoc test). (C) The expression of p21 was determined by Western blot in total lysates from HUVEC. A representative blot is shown on top of each graph, where beta -Actin was employed as loading control (n= 6-8; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1 beta -treated cells, by two-way ANOVA and Tukey post-hoc test). All data are shown as mean ± SEM. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35111374), licensed under a CC-BY license. Not internally tested by R&D Systems.

Protein expression of inflammasome NLRP3 in PBMCs in the presence of Convulxin. Western blot of NLRP3, Pro-Caspase-1, Active Caspase-1, IL-1 beta , and beta -actin (control) using 1 × 107 PBMCs stimulated RPMI (negative control), LPS (1 μg/mL; positive control) or CVX (5 and 10 μg/mL) for 3 h. (A) Figure representative of one experiment of three independent experiments. Relative immunoreactivity analysis (fold of beta -actin) of the western blots from NLRP3, Pro-Caspase-1, Active Caspase-1, IL-1 beta (B). IL-1 beta release was also quantified in PBMCs pretreated with N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (30 µM), Ac-YVAD-cmk (50 µM) (C), MCC950 (10 µM) (D) for 30 min and after incubated with RPMI (negative control) or CVX (10 µg/mL). IL-1 beta was also quantified in PBMCs pretreated with Apocynin (300 µM) and Rotenone (10 µM) for 30 min and after incubated with RPMI (negative control) or CVX (10 µg/mL) (E). Results were expressed in pg/mL of released cytokines and represent the mean ± S.E.M of 3 donors. Values are mean S.E.M. from 3 donors. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to negative control (data were presented with ANOVA followed by Dunnett post-test). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35304541), licensed under a CC-BY license. Not internally tested by R&D Systems.

149-01 targets Cys409 of NLRP3. HEK-293T cells transfected with Flag-tagged NACHT (A), LRR (B), PYD (C), or NACHT (C409A) (D) were lysed and the cell lysates incubated overnight with or without 149-01 (80 μM) before pronase digestion were detected by western blot. (E) The interactions between NEK7 and WT or mutant NLRP3 in HEK-293T cells treated with or without 149-01 (3 μM) were evaluated by IP and western blot. (F,G)Nlrp3−/− BMDMs reconstituted with mouse WT or mutant NLRP3 were first primed with LPS, then treated with or without 149-01 (600 nM) for 30 min, lastly stimulated with nigericin. (F) IL-1 beta releases in supernatants were detected by ELISA. (G) Active IL-1 beta and p20 in supernatants and NLRP3, pro-IL-1 beta , pro-caspase-1 and beta -actin in cell lysates were measured by western blot. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m (n = 6) (F), or are representative of three independent experiments (A–E,G). One-way ANOVA was applied to calculate statistical significance: ***p <.001, NS, not significant. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35250572), licensed under a CC-BY license. Not internally tested by R&D Systems.

AMPK activation restores A beta 42‐induced mitochondrial hyperfission and inflammasome activation in LPS‐primed microglia. LPS‐primed microglia were pretreated with AICAR (1 mM) for 1 h and then treated with A beta 42 for 24 h. (a–e) Representative immunoblot (a) and densitometric quantifications (b–e) of the protein levels of pAMPK, AMPK, pSyk, Syk, Drp1, pro‐IL‐1 beta , and beta ‐actin in total cell lysate. (f–g) MitoSOX (red) and DAPI (blue) staining and quantification of MitoSOX fluorescence intensity. Scale bar: 10 μm. (h) Immunostaining of ASC (red) and DAPI (blue). Scale bar: 10 μm. (i) Percentages of microglia containing ASC foci were counted. Data are shown as mean ± SEM from at least three independent experiments and are analyzed by one‐way ANOVA and Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35474599), licensed under a CC-BY license. Not internally tested by R&D Systems.

Ang-(1-7) attenuates the induction and activation of the NLRP3 inflammasome mediated by IL-1 beta in HUVEC. The expression of NLRP3 inflammasome components (A) NLRP3 and (B) pro-IL-1 beta was determined by Western blot in total lysates from HUVEC stimulated for 18-24 h with IL-1 beta (2.5 ng/ml) in presence or absence of Ang-(1-7) (100 nM). Representative blots are shown on top of the corresponding graphs, where beta -Actin was employed as loading control (n= 6-8; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1 beta -treated cells, by two-way ANOVA and Tukey post-hoc test). (C) The NLRP3 inflammasome activation was determined by the detection of ASC specks by indirect immunofluorescence. The percentage of ASC specks per culture field was quantified under a fluorescence microscope (n= 3-4; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1 beta -treated cells, by two-way ANOVA and Tukey post-hoc test). (D) Confocal representative photographs (630X magnification) where white arrowheads show the location of toroidal-shaped ASC specks as revealed by a specific antibody against ASC (red), while cell nuclei were counterstained with DAPI (blue). (E) levels of mature IL-1 beta were measured in total cell lysates by Western blot, and also (F) detected in the supernatants of treated cells by ELISA (n= 3-4; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1 beta -treated cells, by two-way ANOVA and Tukey post-hoc test). All data are shown as mean ± SEM and expressed as percentage of IL-1 beta -induced levels. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35111374), licensed under a CC-BY license. Not internally tested by R&D Systems.

miR-1208 is the key miRNA bind to METTL3 in EVs to exert anti-inflammatory effects. A. Western blot analysis of Ago-2 expression in hucMSCs lysates. B. Western blot analysis of IL-1 beta and cleaved caspase-1 levels in culture SN and pro-IL-1 beta , pro-caspase-1, NLRP3, and METTL3 levels in macrophage cell lysates in each group. C. Heat map of miRNA levels in hucMSCs-EVs (n = 3, Student’s t test). D. Four most significantly upregulated miRNAs in hucMSCs-EVs. E. The target sequence of miR-1208 estimated within 3′-UTR of METTL3. F. Western blot analysis of IL-1 beta and cleaved caspase-1 levels in culture SN and pro-IL-1 beta , pro-caspase-1, NLRP3, and METTL3 levels in macrophage cell lysates in each group. G. Luciferase reporter assay showing that METTL3 is the target gene for miR-1208 (n = 3, Student’s t test) Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35842714), licensed under a CC-BY license. Not internally tested by R&D Systems.

Multiple TCAs specifically induce the NLRP3 inflammasome activation by triggering upstream signaling events. a The structures of these antidepressants. b LPS-primed BMDMs were stimulated with protriptyline, amitriptyline, nortriptyline, imipramine, doxepin, clomipramine, agomelatine, mirtazapine, and phenothiazine. Western blot assessed the expression of caspase-1 and IL-1 beta in SN and GSDMD cleavage in WCL. c BMDMs were incubated with LPS and MCC950 and then treated with imipramine, amitriptyline, protriptyline and nortriptyline. The expressions of IL-1 beta and caspase-1 in SN as well as GSDMD cleavage in WCL were evaluated using western blotting. dNlrp3−/− BMDMs were incubated with LPS followed by imipramine, amitriptyline, protriptyline and nortriptyline stimulation. The expression of IL-1 beta and caspase-1 in SN and GSDMD cleavage in WCL were measured by western blotting. e BMDMs or hepatocytes were pretreated with LPS and MCC950 and then treated with nortriptyline, western blot was used to measure the expression of caspase-1 in SN. f BMDMs or Kupffer cells were incubated with LPS and MCC950 followed by nortriptyline treatment. The expression of caspase-1 in SN were assessed by western blot Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37231437), licensed under a CC-BY license. Not internally tested by R&D Systems.

IL‐1 beta secretion occurs independently of pore formationWild‐type and GsdmD−/− HoxB8 macrophages were treated as indicated for 16 h. Supernatants were analyzed for IL‐1 beta secretion by ELISA.HoxB8 macrophages were seeded in an 8‐well Ibidi chamber and treated for 14 h with LPS and GM‐CSF. Cells were fixed and stained for gasdermin D (green), IL‐1 beta (red), and DAPI (blue). Images were taken at the Zeiss LSM880 Confocal microscope.HoxB8 macrophages were co‐stimulated with three different PEG 3 mM for 16 h as indicated. Supernatants were analyzed for IL‐1 beta secretion by ELISA.HoxB8 macrophages were seeded in a 96‐well microplate and treated as indicated. Before treatment, SYTOX green was added and cells were imaged in an IncuCyte incubator microscope every hour for 16 h. Shown is the SYTOX green signal normalized to the total cell area.Wild‐type and GsdmD−/− HoxB8 macrophages were treated as indicated for 16 h. Proteins were extracted and analyzed for IL‐1 beta ubiquitination by western blot.Data information: n ≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one‐way ANOVA with multiple comparisons, or two‐way ANOVA to compare across genotypes. P‐values are shown. Microscopy pictures and westerns are representative images from three biological replicates. Scale bars in microscopy images represent 10 µm.Source data are available online for this figure. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35695080), licensed under a CC-BY license. Not internally tested by R&D Systems.

NLRP3 inflammasome activation in MSC-mediated mandibular healing. Two-month-old WT or Prx1-Cre/ROSAnTnG mice used, as specified in the figure legends. Mice received mandibular osteotomy surgery & sacrificed at various time points during healing, N=4. (F-H) M-MSCs from WT mice cultured & stimulated with or without 10 μg/ml LPS ± 5 mM ATP during culture, as indicated in the figures. (F) WB of cell lysates & supernatants from M-MSCs cultures. Image collected & cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/36778113), licensed under a CC-BY license. Not internally tested by R&D Systems.

GM‐CSF induces NLRP3‐dependent IL‐1 beta secretionBMDMM were treated as indicated with either 60 ng/ml ultra‐pure (UP) LPS, 50 ng/ml human‐derived recombinant GM‐CSF, 40 µM WEHD‐FMK for 16 h, or 5 µM nigericin for the last hour. Supernatants were then analyzed for IL‐1 beta secretion by ELISA.HoxB8 macrophages were treated with the indicated dilutions of GM‐CSF using supernatants from GM‐CSF‐expressing cells. Shown are ELISA analysis of IL‐1 beta secretion in supernatants and western blots indicating IL‐1 beta expression in lysates.HoxB8 macrophages of the indicated genotypes were treated as indicated with 60 ng/ml UP‐LPS, 1% GM‐CSF containing supernatant (approximately 2 ng/ml GM‐CSF), 40 µM WEHD.FMK, and 0.5 µM CRT‐0066101 for 16 h; 5 µM nigericin was added for the last hour. Supernatants were analyzed by ELISA for IL‐1 beta . Inlay panels show western blots for levels of IL‐1 beta expression in the indicated knockouts.HoxB8 cells were treated with 60 ng/ml of UP‐LPS, 1% GM‐CSF supernatant, 50 mM KCl, and 5 µM nigericin as indicated for 16 h before supernatants were analyzed for IL‐1 beta by ELISA. Data information: n ≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one‐way ANOVA with multiple comparisons or two‐way ANOVA to compare across genotypes. P‐values are shown. Source data are available online for this figure. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35695080), licensed under a CC-BY license. Not internally tested by R&D Systems.

A 1-hydroxy phenothiazinium-based small molecule inhibits OMV-mediated cytotoxicity and cytokine release in vitro.A Scheme showing the screening strategy to identify potent inhibitors of caspase-11 signaling; B Drug-like properties of PHZ-OH; C Cytotoxicity and IL-1 alpha secretion of wild type (WT), Casp11−/−, Gsdmd−/−, or Nlrp3−/− mouse peritoneal macrophages under a treatment of PHZ-OH (5 μM), and/or a challenge of OMV (10 μg/mL) or E. coli (a MOI of 10) for 16 h; D Caspase-11 mRNA level and cytotoxicity of macrophages primed using LPS (1 μg/mL), Pam3cSK4 (1 μg/mL) or Poly(I:C) (1 μg/mL) for 3 h and challenged with OMV (10 μg/mL) or E. coli (a MOI of 10) for 16 h in the presence or absence of PHZ-OH (5 μM); E TLR4-dependent cytokines (TNF alpha and IL-6) released from WT or Casp11−/− macrophages under a treatment of saline or PHZ-OH (5 μM), and/or a challenge of OMV (10 μg/mL) for 16 h; F Western blots for IL-1 alpha , IL-1 beta and caspase-11 expression and GSDMD cleavage in saline-treated or OMV-challenged macrophages under a treatment of saline or different doses of PHZ-OH (P, 5 or 10 μM); G–J LDH (G or I) and IL-1 alpha as well as IL-1 beta (H or J) released from mouse peritoneal macrophages (WT and Casp11−/−) or THP-1 cells (Transfected with control or caspase-4 siRNA), respectively, in the presence of different concentrations of PHZ-OH (1, 2, 4 μM) and challenged with OMV (10 μg/mL) for 16 h. Data are shown as mean ± SEM of three independent experiments. *P < 0.05. ns, not significant. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35982051), licensed under a CC-BY license. Not internally tested by R&D Systems.

Apilimod mediates inflammasome activation through mitochondrial damage and reactive oxygen species production. (A) Analysis by confocal microscopy of LPS-primed BMDMs stimulated with nigericin or apilimod and subsequently stained with MitoTracker Red (50 nM) follow by the DNA-binding dye DAPI (blue). Scale bar, 20 μm. (B) Analysis by FACS of THP1 cells stimulated with nigericin or apilimod and subsequently stained with MitoTracker deep red (50 nM) and MitoTracker green (50 nM). (C, D) Analysis by confocal microscopy and FACS of BMDMs stimulated with nigericin or apilimod and subsequently stained with MitoSOX Red (1 μM). Scale bar, 20 μm. (E, F) LPS-primed BMDMs were pretreated with a ROS inhibitor (MnTBAP, 150 μM) for 30 min and then stimulated with apilimod for 2 hours. (E) ELISA of IL-1 beta in culture supernatants. (F) Western blot of IL-1 beta , cleaved caspase-1 in supernatants (SN) and pro-IL-1 beta , pro-caspase-1, beta –actin in cell lysate (Input). The data are from three independent experiments with biological duplicates in each and are shown as the mean ± SEM values (n = 3 or 4) or are representative of three independent experiments. One-way ANOVA was applied to calculate statistical significance: ***P < 0.001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37359517), licensed under a CC-BY license. Not internally tested by R&D Systems.

Apilimod damage the mitochondria through inducing lysosomal calcium flux. (A, B) BMDMs were first primed with LPS for 3 hours, then pretreated with BAPTA-AM (25 μM, 50 μM) for 30 minutes, and stimulated with apilimod for 2 hours. (A) Fluo-4 AM (5 μM) staining of BMDMs after apilimod treatment, FACS analysis was conducted to show changes in cell calcium flux. (B) ELISA of IL-1 beta in culture supernatants. (C–E) LPS-primed BMDMs pretreated with ML-SI3 for 30 min and then stimulated with apilimod. (C) Fluo-4 AM staining of BMDMs after apilimod treatment, FACS analysis was conducted to show changes in cell calcium flux. (D) ELISA of IL-1 beta in culture supernatants.(E) Western blot of IL-1 beta , cleaved caspase-1 in supernatants (SN) and pro-IL-1 beta , pro-caspase-1 (pro-casp1), beta –actin in cell lysate. (F, G) BMDMs transfected with siRNA against Trpml1 or control siRNA were primed with LPS for 3 h and were then treated with apilimod or nigericin. The supernatant of the cell culture was collected. (F)Trpml1 mRNA in BMDMs. (G) ELISA of IL-1 beta in the supernatants of BMDMs. (H) LPS-primed THP-1 cells were pretreated with BAPTA-AM (50 μM) or ML-SI3 (40 μM) for 30 minutes, and stimulated with apilimod for 2 hours. subsequently stained with MitoTracker deep red (50 nM) and MitoTracker green (50 nM). Analysis by FACS of THP1 cells. The data are from three independent experiments with biological duplicates in each and are shown as the mean ± SEM values (n = 3) or are representative of three independent experiments. One-way ANOVA was applied to calculate statistical significance: **P < 0.01; ***P < 0.001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37359517), licensed under a CC-BY license. Not internally tested by R&D Systems.

Apilimod induces caspase-1 activation and IL-1 beta secretion. (A–E) BMDMs were first primed with LPS for 3 h, then stimulated with doses of apilimod (2.5-7.5 μM) for 2 hours. The supernatant of the cell culture was collected. (A) ELISA of IL-1 beta releases in culture supernatants. (B) Immunoblot analysis of Active IL-1 beta and p20 (cleaved caspase-1) in supernatants (SN) and pro-IL-1 beta , pro-caspase-1 (pro-casp1) and beta -actin in cell lysates (Input). (C) The release of LDH in supernatants (D) TNF-alpha and (E) IL-6 secretion levels in supernatants were detected by ELISA. (F) Immunoblot analysis of cleaved IL-1 beta and caspase-1 (p20) in culture supernatants (SN) and inactive precursor molecule (pro– IL-1 beta , pro–caspase-1(pro-casp1) in cell lysates (Input) of PMA-differentiated THP-1 cells stimulated with different doses of apilimod. (G) IL-1 beta secretion levels of PBMCs in supernatants were determined by ELISA. The data are from three independent experiments with biological duplicates in each and are shown as the mean ± SEM values (n = 4 or 3) or are representative of three independent experiments. One-way ANOVA was applied to calculate statistical significance: ***P < 0.001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37359517), licensed under a CC-BY license. Not internally tested by R&D Systems.

MafB knockdown promotes the activation of the NLRP3 inflammasome.a–c PMs were transfected with 50 nM control or MafB siRNAs. Forty-eight hours after transfection, were treated with 100 ng/ml LPS for 3 h. were then washed & stimulated with or without ATP (5 mM), Nigericin (5 uM) or MSU (1 mM) in serum-free media for an additional 1 h. Levels of the supernatant IL-1 beta (a) & IL-18 (b) were determined by ELISA. Levels of the supernatant LDH (c) were determined by LDH assay. d PMs were transfected, primed & stimulated with the NLRP3 agonists as in (a). Supernatant proteins were precipitated by TCA & combined with the respective cellular extracts. Levels of the indicated proteins were determined by WBting. Vertically stacked bands originate either from a single membrane or from a replicate membrane, with the same sample loadings. e, f PMs were transfected with control siRNAs or MafB siRNAs. Forty-eight hours after transfection, were primed with LPS for 3 h, followed by stimulation with or without Nigericin for 1 h. were then fixed with 4% PFA & subjected to immunofluorescence staining for ASC. Scale bars: 100 μm (e). ASC specks in the were quantified (f). n = 3–8. g, h J774A.1 macrophages were transfected with control siRNAs or MafB siRNAs. 48 h after transfection, were primed with LPS for 4 h, followed by stimulation with or without ATP (5 mM) or Nigericin (10 μM) for 1 h. Levels of the supernatant IL-1 beta & LDH were determined (g). Levels of the indicated proteins in the supernatants & were determined by WBting (h). Vertically stacked bands originate either from a single membrane or from a replicate membrane, with the same sample loadings. Mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001. Representative of three independent experiments. LDH lactate dehydrogenase. Image collected & cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37845329), licensed under a CC-BY license. Not internally tested by R&D Systems.

NPC86 reduces IL-1 beta protein levels. (a) An automated Western blot analysis using JESS was performed on adipose tissue from DIOD and DIOD + NPC86 (500 ng/kg b.w.) mice using antibodies against IL-1 beta and GAPDH. Representative blots and quantification are displayed. A statistical analysis was performed in GraphPad Prism using an unpaired t-test (n = 3 per group). The data are presented as mean ± SEM, with statistical significance indicated as *** p < 0.001. (b) The serum concentrations of IL-1 beta , GM-CSF, G-CSF, and MCP-1 were measured by ELISA in DIOD and DIOD + NPC86 (500 ng/kg and 1 μg/kg b.w.) mice. A statistical analysis was performed in GraphPad Prism using an unpaired t-test (n = 3 per group). The data are presented as mean ± SEM, with statistical significance indicated as * p < 0.05, ** p < 0.01. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/40332318), licensed under a CC-BY license. Not internally tested by R&D Systems.

AP specifically reduces NLRP3 inflammasome activation. (a,b) Cell viability assays of J774A.1 cells (a) and differentiated THP-1 cells (b) treated with AP (10 μg/mL, 50 μg/mL, 100 μg/mL) for 2 h. Cell viability was analyzed by EZ-CYTOX, measured at a wavelength of 450 nm. Ns not significant. (c–e) Western blots of IL-1 beta in the supernatants (Sup), pro-IL-1 beta and alpha -tubulin in the soluble lysates (Lys) of treated cells. LPS-primed J774A.1 cells were treated with AP for 2 h and activated for 30 min with nigericin (10 μM) (c) or ATP (5 mM) (d) with or without MCC950, differentiated, and LPS-primed THP-1 cells were treated with AP for 2 h and activated for 30 min with nigericin (10 μM) with or without MCC950 (e). (f,g) LPS-primed J774A.1 cells were treated with AP for 2 h and activated for 3 h with transfection of dsDNA (2 μg/mL) (f) or flagellin (1.25 μg/mL) (g). Image collected and cropped by CiteAb from the following open publication (//www.nature.com/articles/s41598-024-55651-z), licensed under a CC-BY license. Not internally tested by R&D Systems.

The CR3-Als3 complex at the F-actin cuff promotes Syk phosphorylation and inflammasome activation. A Immunoblot analysis of p-Syk from C57BL/6 and CD18−/− M0 BMDM (without GM-CSF priming) lysates after stimulation with C. albicans hyphae for indicated periods. Total Syk served as a loading control. b WT M0 BMDMs were pretreated with anti-CD11b blocking antibody (M1/70, 10 μg/mL) for 30 min before stimulation with C. albicans hyphae. Cell lysates were subjected to immunoblot analysis of p-Syk and total Syk. IgG2b, kappa was used as an isotype control. c Immunoblot analysis of p-Syk and total Syk from C57BL/6 or CD18−/− M0 BMDMs after WT, als1 delta / delta als3 delta / delta , or als1 delta / delta als3S170Y hyphal infection. d Immunoblot analysis of p-Syk from C57BL/6 and CD18−/− M0 BMDM (without GM-CSF priming) lysates after stimulation with 15 µg ml-1 Als3 or Als3S170Y for indicated periods. Total Syk served as a loading control. e Immunofluorescence images of M0 BMDMs after stimulation with C. albicans hyphae expressing TagBFP for 20 min. p-Tyr, CD11b, and F-actin were analyzed. Scale bars, 7.5 µm. f Immunofluorescence images of M1 BMDMs after infection with C. albicans hyphae expressing TagBFP for 20 min. p-Syk, CD11b, and Dectin-2 were analyzed. Scale bar, 7.5 µm. g Immunoblot analysis of caspase-1 and IL-1 beta after infection with C. albicans hyphae in BMDMs with or without Cyto D and Lat B. Hyphae were induced in RMPI medium a–c, e, f and SC medium plus GlcNAc g. Each experiment was repeated three times independently. Data in g are presented as mean ± SEM. P values in g were determined by one-way ANOVA (Tukey’s multiple comparisons test). ns not significant. Source data are provided as a Source Data file. Image collected and cropped by CiteAb from the following open publication (//www.nature.com/articles/s41467-024-48093-8), licensed under a CC-BY license. Not internally tested by R&D Systems.

Inhibition of Clec7a effectively mitigates microglial synaptic phagocytosis and neuroinflammation in PS19 mice. (A) Representative Western blots of indicated proteins in the ventral hippocampal lysates from 6-month-old WT mice and Laminarin-administered PS19 mice. (B) Quantitation of Clec7a, P-SYK, NLRP3, ASC, Procaspase-1, and IL-1 beta proteins in (A) (n = 6 mice per group, unpaired Student’s t test). (C) Representative Western blots of indicated proteins in the ventral hippocampal lysates from 6-months old WT mice and Laminarin-administered PS19 mice. (D,E) Quantitation of PSD95 (D) and synaptophysin (E) proteins (n = 6 mice per group, unpaired Student’s t test). (F) Confocal images of synaptic puncta engulfed by microglia showing the colocalization of PSD95 (green) with CD68 (grays) within Iba1+ microglia (red) in the DG regions of hippocampal tissue from WT mice and Laminarin-administered PS19 mice. Scale bars, 5 μm. (G) Quantitation of CD68 volume. (H) Quantitation of PSD95 engulfment within CD68+ microglial structures (n = 6 mice per group, one-way ANOVA test). * p < 0.05; ** p < 0.01; *** p < 0.001. Data are presented as mean ± SD. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/40243488), licensed under a CC-BY license. Not internally tested by R&D Systems.

Pyroptosis is the primary mode of AEC death induced by cadmium. MLE12 cells were treated with different concentrations of CdCl2 for 24 h. Cell morphology was photographed through a microscope (A, bar = 100 μm). Cell viability was detected by CCK8 assay (B, n = 5). The LDH activity of the cell culture supernatant was assayed (C, n = 3). The Pan-caspase inhibitor (ZVAD-FMK) but not the necroptosis or ferroptosis inhibitor restored cell viability within 24 h of CdCl2 incubation (D, n = 5). Cell morphology was photographed through a microscope (E, bar = 100 μm). The expressions of SPC, NLRP3, pro-Caspase1, Caspase1 p20, IL-1 beta p17, and GSDMD-N protein in MLE12 cells were detected using western blot (F-G, n = 3). The fluorescence intensity of GSDMD was detected by immunofluorescence (H, bar = 5 μm). Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. Tukey’s test was used as a post hoc test for pairwise comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001 Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/39587603), licensed under a CC-BY license. Not internally tested by R&D Systems.

Hectd3 deficiency promotes the activation of NLRP3 inflammasome.A–H BMDMs isolated from Hectd3+/+ and Hectd3−/− mice were primed with LPS and stimulated with different secondary signals, including ATP and MSU. Supernatant IL-1 beta , IL-6 and TNF-alpha were analyzed using ELISA, n = 3 per group (A–C). Supernatants (SN) and cell extracts lysate (Lys) were analyzed using immunoblot assays (D). Cell death were detected by CCK8 asssay, n = 4 per group (E). Supernatants were collected to measure cell death by LDH release assay, n = 3 per group (F). PI staining was detected by fluorescence and the quantification of PI-positive cells is shown, n = 3 per group (G). The cleavage of GSDMD were detected using immunoblot assays (H); I, J. HEK293T cells were transfected with Flag-NLRP3, Myc-ASC, Flag-pro-caspase-1, Flag-pro-IL-1 beta plasmids as well as si-RNA of HECTD3 and stimulated with 2.5 mM ATP. Supernatants (SN) and cell extracts lysate (Lys) were analyzed using immunoblot assays (I); Supernatant IL-1 beta were analyzed using ELISA, n = 3 per group (J); K BMDMs isolated from Hectd3+/+ and Hectd3−/− mice were treated with 200 ng/mL LPS for 15 or 30 min, cell extracts were analyzed with immunoblot assays. Data are the mean ± SEM, ns (non-significant), P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001, two-tailed unpaired Student’s t test was used. Image collected and cropped by CiteAb from the following open publication (//www.nature.com/articles/s41419-024-06473-4), licensed under a CC-BY license. Not internally tested by R&D Systems.

AP does not affect the NF-kappa B signaling pathway. (a) 293 T cells were transfected with the pGL4.32 luciferase reporter vector and treated with TNF-alpha (20 ng/mL) for 5 h with or without AP. Luciferase activity was analyzed using the Promega Bright-Glo™ Luciferase Assay System. (b) LPS-primed J774A.1 cells were treated with AP for 2 h and activated for 30 min with nigericin (10 μM). IL-1 beta in the supernatants (Sup), phospho-NF-kappa B and NF-kappa B in the soluble lysates (Lys) were analyzed using a Western blot. Image collected and cropped by CiteAb from the following open publication (//www.nature.com/articles/s41598-024-55651-z), licensed under a CC-BY license. Not internally tested by R&D Systems.

Neutrophil recruitment contributes to BBB disruption following peripheral inflammatory stimulation.a Representative immunoblots detecting pro-IL-1 beta , IL-1 beta , and NLRP3 in brain lysates of WT mice at different time points (0, 1 ~ 6 h) after the second LPS injection. b, c Quantification of brain-infiltrating myeloid cells or neutrophils in the brain of WT mice upon PBS or repeated LPS injection at different time points by flow cytometry. (1 ~ 6 h post second LPS injection, n = 5, PBS, 4 h; 6, 3 h; 4, all other group). d Violin plot showing chemokine receptor gene expression in neutrophils of all groups. e Circle plot visualizing cell-cell communication in the CXCL signaling pathways for each experimental group. The plot displays interactions among two cell types in the PBS group and 5 cell types (Endo Endothelial cell, Peri Pericyte, Astro Astrocyte, Micro Microglia, Neutro Neutrophil) in the LPS x2 group. The width and color of arrows represent the probability of interaction. f Quantification of Evans blue depositions in the WT mouse brains upon repeated LPS injection pretreated with IgG (n = 4) or anti-Ly6G (n = 6) antibodies (50 μg, i.p.) 1 h before 1st LPS injection. g Quantification of Evans blue depositions in the WT mouse brains upon repeated LPS injection in the presence of vehicle (n = 6) or anti-CXCR2 antagonist SB225002 (n = 8) treatment (2 mg/kg, i.p.). h Quantification of Evans Blue extravasation into the brain followed by LPS injections in WT and Ccr2−/− mice. n = 5 (WT) or 3 (Ccr2−/−) mice. Error bars, s.e.m. One-way ANOVA with Dunnett post hoc test (b, c) and unpaired two-sided Student’s t test (f, g, h). Source data are provided as a Source Data file. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/39814731), licensed under a CC-BY license. Not internally tested by R&D Systems.

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_lightbox">

Recombinant Mouse IL‑1 beta /IL‑1F2 (Catalog # <a class=

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_thumb" />

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_lightbox">

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_thumb" />

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_lightbox">

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_thumb" />

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_lightbox">

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_thumb" />

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_lightbox">

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_thumb" />

Reactivity:

Mu

Species Glossary

Reactivity: Mu

Applications:

, , , ,

Applications: , , , ,

Host:

Goat Polyclonal

Host: Goat Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	AF-401-NAU-100UG
Alexa Fluor 405	AF-401-NAV-100UG
Alexa Fluor 488	AF-401-NAG-100UG
Alexa Fluor 532	AF-401-NAX-100UG
Alexa Fluor 594	AF-401-NAT-100UG
Alexa Fluor 647	AF-401-NAR-100UG
Alexa Fluor 700	AF-401-NAN-100UG
Alexa Fluor 750	AF-401-NAS-100UG
Alexa Fluor Plus 405	AF-401-NAAFP405
Alexa Fluor Plus 488	AF-401-NAAFP488
Alexa Fluor Plus 555	AF-401-NAAFP555
Alexa Fluor Plus 594	AF-401-NAAFP594
Alexa Fluor Plus 647	AF-401-NAAFP647
Alexa Fluor Plus 680	AF-401-NAAFP680
Allophycocyanin	AF-401-NAA
Biotin	BAF401
CoraFluor 1	AF-401-NACL1
DyLight 350	AF-401-NAD
DyLight 405	AF-401-NAE
DyLight 488	AF-401-NAK
DyLight 550	AF-401-NAL
DyLight 594	AF-401-NAM
DyLight 650	AF-401-NAW
DyLight 680	AF-401-NAY
DyLight 755	AF-401-NAZ
FITC	AF-401-NAF
HRP	AF-401-NAH
Janelia Fluor 525	AF-401-NAJF525
Janelia Fluor 549	AF-401-NAI
Janelia Fluor 646	AF-401-NAJ
Janelia Fluor 669	AF-401-NAJF669
PE	AF-401-NAP
PerCP	AF-401-NAC
mFluor Violet 450 SE	AF-401-NAMFV450
mFluor Violet 500 SE	AF-401-NAMFV500
mFluor Violet 610 SE	AF-401-NAMFV610

Available Formulations

Formulation	Catalog #	Availability	Price
Carrier Free	AF-401-SP

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AB-105-C

Rabbit IgG Isotype Control [Unconjugated]

(2 Reviews)

(180 Publications)

1 mg

AB-105-C

MAB58891, filled histogram) or Rabbit IgG Isotype Control Antibody (Catalog # AB-105-C, open histogram), followed by Allophycocyanin-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0111). View our protocol for Staining Membrane-associated Proteins." class="big_lightbox">

HepG2 human hepatocellular carcinoma cell line was stained with Rabbit Anti-Human Klotho beta Monoclonal Antibody (Catalog # <a class=

MAB58891, filled histogram) or Rabbit IgG Isotype Control Antibody (Catalog # AB-105-C, open histogram), followed by Allophycocyanin-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0111). View our protocol for Staining Membrane-associated Proteins." alt="HepG2 human hepatocellular carcinoma cell line was stained with Rabbit Anti-Human Klotho beta Monoclonal Antibody (Catalog # MAB58891, filled histogram) or Rabbit IgG Isotype Control Antibody (Catalog # AB-105-C, open histogram), followed by Allophycocyanin-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0111). View our protocol for Staining Membrane-associated Proteins." class="big_thumb" />

<b>Biological Strategies Validation. </b>pSTAT5, but not NS1, interacts with the MCM complex.(A) Immunoprecipitation (IP) assay. Cell lysates of NS1Flag-expressing UT7/Epo-S1 cells were prepared for pull-down assays with either anti-Flag-conjugated beads or control beads. Immunoprecipitated proteins were examined for the presence of MCM2 by Western blotting. Blots were reprobed with rabbit anti-pSTAT5(Y694), anti-E2F5, and anti-Flag antibodies. Detection of E2F5 was used as a positive control for NS1 IP. (B) Co-IP assay. UT7/Epo-S1 cells were collected, washed, and lysed with RIPA buffer. After centrifugation, the supernatant was incubated with either rabbit anti-pSTAT5(Y694) or control IgG antibody. Immunoprecipitated proteins were blotted for the presence of the MCM complex with an anti-MCM5 antibody and for pSTAT5 with rabbit anti-pSTAT5(Y694). (C) Reverse Co-IP assay. Reverse Co-IP was performed with an anti-MCM5 antibody. Immunoprecipitated proteins were examined for pSTAT5, MCM2, and MCM5, respectively. (D) Co-IP of lysates treated with DNase. UT7/Epo-S1 cell lysates, either treated or untreated with DNase (750 units of Benzonase) were incubated with anti-pSTAT5(Y694) or control IgG antibodies for Co-IP assay, and immunoprecipitated proteins were examined for MCM2 by Western blot analysis. (E-H) Immunofluorescence analysis. (E&F) Mock- or B19V-infected CD36+ EPCs were co-stained with rabbit anti-STAT5 and mouse anti-MCM2 antibodies, followed by (E) incubation with respective secondary antibodies, or by (F) proximal ligation assay, which produces amplified signal for labeled molecules in close proximity. (G) CD36+ EPCs were incubated with either DMSO or pimozide (at 30 μM) for 2 days. And then the cells were co-stained with rabbit anti-STAT5 and mouse anti-MCM2 antibodies for proximity ligation assay. (H) Infected EPCs were stained with an anti-capsid antibody. Confocal images were taken with an Eclipse C1 Plus (Nikon) microscope at 100 × magnification. Image collected and cropped by CiteAb from the f

$Protein-protein interaction between ANO1 and NCX3. (A) Co- IP of ANO1 and NCX3 from small intestine smooth muscle membrane fraction. Immunoprecipitates (IP) were obtained by elution with low pH buffer, and immunoblotting (IB) was performed using Wes with anti-ANO1 and anti-NCX3 antibodies. Lanes: 1, protein standards; 2, ANO1 in membrane fraction (2.5 mg); 3, ANO1 in ANO1 immunoprecipitate (5 ml); 4, ANO1 in non-immune rabbit IgG IP; 5, NCX3 in membrane fraction (1 mg); 6, NCX3 in NCX3 immunoprecipitate (5 ml); 7, NCX3 in non-immune rabbit IgG IP; 8, NCX3 in Ano1 immunoprecipitate (5 ml); 9, ANO1 in NCX3 immunoprecipitate (5 ml) n = 4. (B) Association between ANO1 and NCX3 occurs in situ in ICC, as demonstrated by PLA. ICC from small intestine (green, left panel) were exposed to the rabbit anti-ANO1 and goat anti-NCX3 antibodies, followed by the Duolink minus-anti rabbit IgG and plus-anti goat IgG secondary antibodies and the ligation and amplification reactions. For the PLA negative control, ICC were exposed to rabbit anti-ANO1 and mouse anti-myosin regulatory light chain antibodies (upper left panel). PLA-positive red spots were detected by ANO1-NCX3 co-localization (lower left panel). Few red spots were observed in negative controls. Nuclear staining of ICC was achieved with DAPI (blue, left panel). Intact ICC were confirmed by differential interference contrast (DIC) (right panels). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/32256387), licensed under a CC-BY license. Not internally tested by R&D Systems.$

Reactivity:

Rb

Species Glossary

Reactivity: Rb

Applications:

Applications:

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Conjugate:

Unconjugated

Validated by:

Validated by:

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MAB002

Mouse IgG1 Isotype Control (11711) [Unconjugated]

(1 Review)

(432 Publications)

500 μg

MAB002

Human peripheral blood mononuclear cells (PBMCs) were stained with Mouse Anti-Human LILRA6/CD85b Monoclonal Antibody (Catalog # MAB8656, filled histogram) or Mouse IgG1Isotype Control Antibody (Catalog # MAB002, open histogram), followed by Allophycocyanin-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0101B).

EC and PC ICAM-1 expression and effects of CD18 blocking on neutrophil adhesion.ICAM-1 expression on (A) EC or (D) PC monolayers before and after 4 hr IL-1 beta activation was assessed by flow cytometry (IgG labeled control white). ICAM-1 expression on (B,C) EC and (E,F) PC following 4 hr IL-1 beta activation was imaged using confocal microscopy. Scale bars are 20 µm. (G) Neutrophil adhesion inhibition to IL-1 beta activated EC or PC monolayers. Freshly isolated neutrophils were pre-incubated with anti-Mac-1 or anti-LFA-1 antibodies and seeded onto EC or PC monolayers (activated for 4 with IL-1 beta ) in Sykes-Moore chambers and allowed to adhere prior to counting. Bars represent average neutrophil adhesion ± SEM. *P<0.05, when compared to the no block control. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0060025), licensed under a CC-BY license. Not internally tested by R&D Systems.

miR-720 maintains the migratory and invasive properties of MDA-MB-231 cells. MDA-MB-231 cells were transfected with 50 nM anti-miR-720 or anti-miR-negative control (Ctrl) for 48 h. Either RNA was isolated (a) or transfected cells subjected to functional assays (b–e). a miR-720 levels. RNA was subjected to RT-qPCR for miR-720. Control condition (Ctrl) was set to 1.0 (mean ± SD from three independent experiments). b Migration assay. Transfected cells were subjected to a migration assay in a Boyden chamber over a 24-h period. The cells that migrated through the chamber were quantified using crystal violet staining. Ctrl was set to 100 % (mean ± SD from three independent experiments). c Matrigel assay. Transfected cells were grown in Matrigel for 11–15 days and then photographed using a Nikon eclipse TS100 microscope at 10× magnification. Scale bar = 100 μm. Representative images of two independent experiments with similar results are shown (left panels). The number of branched colonies were counted. The count in the control sample was set to 1.0 (right panel). d Transendothelial migration assay. Transfected MDA-MB-231 cells were plated in a Boyden chamber that had been coated with a confluent monolayer of HUVEC cells, and the cancer cells that invaded through the HUVEC layer over a 24-h period were quantified using crystal violet staining. Ctrl value for transendothelial (transendo.) migration was set to 100 % (mean ± SD from three independent experiments) and relative (Rel.) migration values given. e ATP assay. Transfected cells were grown for 24 h and ATP levels were measured using an ATPlite 1Step assay. Ctrl was set to 100 % (mean ± SD from three independent experiments). *P < 0.05, ***P < 0.001, Student’s t test. Not sig. not significant Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27039296), licensed under a CC-BY license. Not internally tested by R&D Systems.

Monoclonal ASM antibody (23A12C3) inhibits pathogenic Th17 cell differentiation and protects neuropathological features in APP/PS1 mice. e Representative flow cytometry plot and graph displaying the calculated percentage and mean fluorescent intensity of Th17 cells (CD4+ IL17A+) differentiated from CD4+ T cell in each group (Control, n = 5; Smpd1ox/ox serum/con IgG ab, n = 10; Smpd1ox/ox serum/23A12C3, n = 8). f Western blotting for p-Stat3, p-JNK, p-AKT, p-mTOR in Th17 cells differentiated from CD4+ T cell in each group (n = 4/group). g ASM activity in plasma (n = 5/group). h Percentage of Th17 cells in the spleen, blood, and brain by flow cytometry (n = 5/group). i Quantification of microglia (Iba-1) and astrocyte (GFAP) (n = 5/group). j mRNA levels of inflammatory markers in the cortex and hippocampus (n = 4/group). k Representative immunofluorescence images and quantification of ThioS (A beta plaques) in cortex and hippocampus. Scale bars, 50 μm. (n = 5/group). l Quantification of 6E10 (n = 5/group). m The results of Morris water maze test (n = 10/group). n, o At probe trial day 11, time spent in target platform (n) and the number of times each animal entered the small target zone (o) were analyzed (n = 10/group). p The results of fear-conditioning test (n = 10/group). d–j, m–p One-way analysis of variance, Tukey’s post hoc test. k, l, n Two-tailed student’s t test. All error bars indicate s.e.m. All in vivo data analysis was done at 9-mo-old mice. Source data are provided as a Source Data file. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/36959217), licensed under a CC-BY license. Not internally tested by R&D Systems.

Reactivity:

Mu

Species Glossary

Reactivity: Mu

Applications:

Applications:

Host:

Mouse Monoclonal

Host: Mouse Monoclonal

Conjugate:

Unconjugated

Available Conjugates:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 405	IC002V
Alexa Fluor 488	IC002G
Alexa Fluor 594	IC002T
Alexa Fluor 647	IC002R
Alexa Fluor 700	IC002N
Alexa Fluor 750	IC002S
Allophycocyanin	IC002A
Biotin	IC002B
Fluorescein	IC002F
PerCP (Peridinin-chlorophyll Protein Complex)	IC002C
Phycoerythrin	IC002P

Available Formulations

Formulation	Catalog #	Availability	Price
Carrier Free	IC002A
Carrier Free	IC002B
Carrier Free	IC002C
Carrier Free	IC002F
Carrier Free	IC002G
Carrier Free	IC002N
Carrier Free	IC002P
Carrier Free	IC002S
Carrier Free	IC002T
Carrier Free	IC002R
Carrier Free	IC002V

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NBP2-24891

Rabbit IgG Isotype Control

(1 Review)

(61 Publications)

0.1 mg

NBP2-24891

Controls for alpha-MSH immunohistochemistry and immunofluorescence. (A) Isotype controls for alpha-MSH were obtained by staining consecutive sections of human and mouse atherosclerotic plaques with alpha-MSH antibody or rabbit polyclonal isotype control antibody. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-018-33523-7) licensed under a CC-BY license..

Flow Cytometry: Rabbit IgG Isotype Control [NBP2-24891] - Intracellular FACS analysis of mouse TLR6 polyclonal antibody (red), rabbit isotype control (green), RAW cells alone (shaded). Two micrograms of antibodies were used. Goat anti-rabbit FITC (Novus, 20302) was used as secondary.

Flow Cytometry: Rabbit IgG Isotype Control [NBP2-24891] - PBMCs fixed and permeabilized using the Novus intracellular staining kit and stained with 1 ug of TLR7-PE conjugate (red) or rabbit IgG-PE conjugate isotype control (NBP2-24983, green). Image using the PE form of this antibody.

Flow Cytometry: Rabbit IgG Isotype Control [NBP2-24891] - Intracellular analysis of TLR1 in human PBMCs (lymphocyte-gated) using this antibody. Shaded histogram represents cells alone, green represents isotype control , red represents TLR1 antibody. TLR intracellular flow kit was used for this test. Image using the Alexa Fluor 488 form of this antibody.

Flow Cytometry: Rabbit IgG Isotype Control [NBP2-24891] - Analysis of TLR7 in Ramos cells using 2 ug of NBP2-24892. Shaded histogram represents FITC-conjugated rabbit IgG isotype control ; red represents anti-TLR7 antibody. Image using the FITC form of this antibody.

Flow Cytometry: Rabbit IgG Isotype Control [NBP2-24891] - FLOW analysis of Mouse CD4+ T cells that were stimulated with anti-CD3/CD28 beads and insulin (1ug/mL) for 5 days in culture media with additional glucose provided. PE conjugated Glut1 antibody stained cells (NB110-39113PE, Orange) were included as positive control which were compared with Rb IgG PE, NBP2-24983 as Isotype Control (Blue) as well as with fluorescence minus one/FMO control (Red) [Image submitted by Verified Customer - see Review for Protocol Details]. Image using the PE form of this antibody.

Immunocytochemistry/ Immunofluorescence: Rabbit IgG Isotype Control [NBP2-24891] - The expression & localization of PRCP in the human atherosclerotic plaque. (A) Immunohistochemical staining of PRCP (brown color) in a carotid endarterectomy sample. Scale bars, 500 µm (left) & 50 µm (right). (B) A consecutive section of the carotid sample was immunofluorescently stained for PRCP (red) & Mac-2 (green), & counterstained with DAPI. Cells that clearly express both PRCP & Mac-2 are indicated by white arrows. Scale bars, 500 µm (left) & 50 µm (right). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30305673), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: Rabbit IgG Isotype Control [NBP2-24891] - alpha -melanocyte stimulating hormone ( alpha -MSH) is expressed by plaque macrophages in human atheroma. (A) alpha -MSH immunostaining (brown color) of carotid endarterectomy sample. Scale bars, 200 µm (left) & 50 µm (right). (B) A consecutive section of the carotid sample was immunofluorescently stained for alpha -MSH (red) & Mac-2 (green), & counterstained with DAPI. Cells that clearly express both alpha -MSH & Mac-2 are indicated by white arrows. Scale bars, 200 µm (left) & 50 µm (right). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30305673), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Rb

Species Glossary

Reactivity: Rb

Applications:

, , , , ,

Applications: , , , , ,

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Alt Catalog#

20304

Alt Catalog# 20304

Conjugate:

Unconjugated

Available Conjugates:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NBP2-24891AF350
Alexa Fluor 405	NBP2-24891AF405
Alexa Fluor 488	NBP2-24982
Alexa Fluor 532	NBP2-24891AF532
Alexa Fluor 594	NBP2-24891AF594
Alexa Fluor 647	NBP2-24891AF647
Alexa Fluor 700	NBP2-24891AF700
Alexa Fluor 750	NBP2-24891AF750
Allophycocyanin	NBP2-24891APC
Allophycocyanin/Cy7	NBP2-24891APCCY7
Biotin	NBP2-24891B
CoraFluor 1	NBP2-24891CL1
DyLight 350	NBP2-24891UV
DyLight 405	NBP2-24891V
DyLight 488	NBP2-24891G
DyLight 550	NBP2-24891R
DyLight 594	NBP2-24891DL594
DyLight 650	NBP2-24891C
DyLight 680	NBP2-24891FR
DyLight 755	NBP2-24891IR
FITC	NBP2-24892
HRP	NBP2-24891H
Janelia Fluor 525	NBP2-24891JF525
Janelia Fluor 549	NBP2-24891JF549
Janelia Fluor 646	NBP2-24891JF646
Janelia Fluor 669	NBP2-24891JF669
PE	NBP2-24983
PE/Cy5.5	NBP2-24891PECY55
PerCP	NBP2-24891PCP
mFluor Violet 450 SE	NBP2-24891MFV450
mFluor Violet 500 SE	NBP2-24891MFV500
mFluor Violet 610 SE	NBP2-24891MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
Azide and BSA Free	NBP2-24893

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NB810-56910

Rabbit IgG Isotype Control

(6 Reviews)

(50 Publications)

10 mg

NB810-56910

Reactivity:

Rb

Species Glossary

Reactivity: Rb

Applications:

, , , , ,

Applications: , , , , ,

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Conjugate:

Unconjugated

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1-001-A

Human IgG Isotype Control [Unconjugated]

(1 Review)

(59 Publications)

1 mg

1-001-A

Activated ADSCs trigger the proliferation and invasion of lung cancer cells by regulating MMP2/9 expression and EMT. a The effects of activated ADSCs on lung cancer cell proliferation were evaluated using the CCK-8 assay. Four lung cancer cell lines were cultured in the presence of ADSC-CM or aADSC-CM, and the optical density at 450 nm was analysed. The cancer cells cultured alone in normal growth medium were used as negative controls. Data from three separate experiments are shown. b The number of lung cancer cells that migrated through 8-μm Transwell membrane pores was counted to determine the changes in the invasive capabilities in response to CM from epidural ADSCs or aADSCs. c MMP2/9, E-cadherin and vimentin expression levels in four lung cancer cell lines treated with ADSC-CM or aADSC-CM were examined by western blotting. Lung cancer cells cultured in an untreated medium served as controls. d Lung cancer cells were treated with neutralizing antibodies against IL-6, isotype controls and aADSC-CM. Lung cancer cell invasion was analysed using a Transwell assay. Lung cancer cells cultured with untreated medium served as negative controls. e Lung cancer cells were treated with neutralizing antibodies against IL-6, isotype controls and aADSC-CM. MMP2/9, E-cadherin and vimentin expression levels in four lung cancer cell lines were analysed using western blotting. Lung cancer cells cultured with untreated medium served as negative controls. ***P < 0.001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/31196220), licensed under a CC-BY license. Not internally tested by R&D Systems.

Reactivity:

Hu

Species Glossary

Reactivity: Hu

Applications:

Applications:

Host:

Human Polyclonal

Host: Human Polyclonal

Conjugate:

Unconjugated

Validated by:

Validated by:

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HAF019

Chicken anti-Goat IgG Secondary Antibody [HRP]

(1 Review)

(46 Publications)

1 mL

HAF019

AF4920) using a 1:1000 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF019). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1." class="big_lightbox">

Western blot shows detection of Goat Anti-Human/Mouse/Rat Carboxylesterase 1/CES1 Polyclonal Antibody (<a class=

AF4920) using a 1:1000 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF019). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1." alt="Western blot shows detection of Goat Anti-Human/Mouse/Rat Carboxylesterase 1/CES1 Polyclonal Antibody (AF4920) using a 1:1000 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF019). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1." class="big_thumb" />

AF2105) followed by 30 min incubation with Donkey Anti-Goat HRP conjugated secondary antibodies (Catalog# HAF019) at 1:20 dilution + DAB chromogen (brown). The tissue was counterstained with Hematoxylin (blue). Control was done by omitting primary antibody." class="big_lightbox">

Detection of Vimentin in human kidney using 5 ug/mL of Goat anti-human Vimentin primary antibody (<a class=

AF2105) followed by 30 min incubation with Donkey Anti-Goat HRP conjugated secondary antibodies (Catalog# HAF019) at 1:20 dilution + DAB chromogen (brown). The tissue was counterstained with Hematoxylin (blue). Control was done by omitting primary antibody." class="big_thumb" />

Reactivity:

Gt

Species Glossary

Reactivity: Gt

Applications:

,

Applications: ,

Host:

Chicken Polyclonal

Host: Chicken Polyclonal

Conjugate:

HRP

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5-001-A

Sheep IgG Isotype Control [Unconjugated]

(1 Review)

(46 Publications)

1 mg

5-001-A

Effects of SDF-1-CXCR4/CXCR7 pathway on MSC chemotaxis in vitro.(A) The chemotaxis in response to SDF-1 alpha (10 ng/ml for 12 h) was performed in the NP-MSCs and HP-MSCs treated with a neutralizing anti-CXCR4 antibody, an anti-CXCR7 antibody, and the respective isotype-matched control antibodies. *P<0.05, vs NP-MSCs; †P<0.05, vs the respective isotype-matched control antibodies. (B) NP-MSCs were transiently overexpressed with CXCR4 using pORF9-mCXCR4 vector or with CXCR7 using pORF9-mCXCR7 vector (n = 6). A negative control empty (pORF9-MCS) vector was used. (C) The transfected cells were subjected to chemotaxis in response to the indicated concentrations of SDF-1 alpha for 12 h. *P<0.05, vs the empty vector. (D and E) Western blot analysis (D) and FCM (E) were performed to determine the intracellular and extracellular expression of both CXCR4 and CXCR7 in the cells treated with or without SDF-1 alpha (50 ng/ml for 60 min). (F) The chemotaxis in response to SDF-1 alpha was performed in the cells treated with or without SDF-1 alpha . Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/22511954), licensed under a CC-BY license. Not internally tested by R&D Systems.

Reactivity:

Sh

Species Glossary

Reactivity: Sh

Applications:

Applications:

Host:

Sheep Polyclonal

Host: Sheep Polyclonal

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 488	IC016G
Alexa Fluor 647	IC016R
Alexa Fluor 700	IC016N-100UG
Allophycocyanin	IC016A
Biotin	BAF020
Fluorescein	IC016F
Phycoerythrin	IC016P

Available Formulations

Formulation	Catalog #	Availability	Price
Carrier Free	IC016N-100UG
Carrier Free	IC016A
Carrier Free	IC016F
Carrier Free	IC016G
Carrier Free	IC016P
Carrier Free	IC016R

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6-001-A

Rat IgG Isotype Control [Unconjugated]

(34 Publications)

1 mg

Reactivity:

Rt

Species Glossary

Reactivity: Rt

Applications:

Applications:

Host:

Rat Polyclonal

Host: Rat Polyclonal

Conjugate:

Unconjugated

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NB7156

Goat anti-Rabbit IgG (H+L) Secondary Antibody

(65 Publications)

1 ml

NB7156

Reactivity:

Rb

Species Glossary

Reactivity: Rb

Applications:

, , , ,

Applications: , , , ,

Host:

Goat Polyclonal

Host: Goat Polyclonal

Conjugate:

Unconjugated

Available Conjugates:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor Plus 405	NB7156AFP405
Alexa Fluor Plus 488	NB7156AFP488
Alexa Fluor Plus 555	NB7156AFP555
Alexa Fluor Plus 594	NB7156AFP594
Alexa Fluor Plus 647	NB7156AFP647
Alexa Fluor Plus 680	NB7156AFP680
Allophycocyanin	NB7156APC
Biotin	NB7158
DyLight 350	NB7156UV
DyLight 405	NB7156V
DyLight 488	NB7156G
DyLight 550	NB7156R
DyLight 594	NB7156DL594
DyLight 650	NB7156C
DyLight 680	NB7156FR
DyLight 755	NB7156IR
FITC	NB7159
HRP	NB7160
Janelia Fluor 525	NB7156JF525
Janelia Fluor 669	NB7156JF669
PE	NB7156PE
PerCP	NB7156PCP

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NBP2-36463

Rabbit IgG Isotype Control

(1 Review)

(8 Publications)

0.5 mg

NBP2-36463

SDS-Page: Rabbit IgG Isotype Control [NBP2-36463] - Lane 1: Non-reduced Rabbit IgG Whole Molecule. Lane 2: 5uL OPAL Pre-stained Marker. Lane 3: Reduced Rabbit IgG Whole Molecule. Load: 1ug per lane. Predicted/Observed size: Non-reduced at 150-170 kDa , Reduced at 55, 25 kDa.

SDS-Page: Rabbit IgG Isotype Control [NBP2-36463] - SDS-Page of Rabbit IgG. Lane 1: Rabbit IgG - Non-Reduced. Lane 2: Rabbit IgG - Reduced. Load: 1.0 ug per lane. Predicted/Observed Size: Non-reduced - 130 kDa, Reduced - 55 and 28 kDa for Rabbit IgG. Other Band(s): None.

SDS-Page: Rabbit IgG Isotype Control [NBP2-36463] - Lane M: 3 uL Opal Prestained Marker. Lane 1: Reduced Rabbit IgG Whole Molecule. Lane 2: Reduced Rabbit IgG F(ab) Fragment. Lane 3: Reduced Rabbit IgG F(c) Fragment. Lane 4: Reduced Rabbit IgM Whole Molecule. Load: 1 ug for F(ab) and F(c); 1.2 ug for IgG and IgM. Predicted/Observed size: IgG at 50 and 25 kDa; F(c) at 25 kDa; F(ab) at 25 kDa; IgM at 70 and 23 kDa. Observed F(c) Fragment migrates slightly higher.

Reactivity:

Rb

Species Glossary

Reactivity: Rb

Applications:

, , , , ,

Applications: , , , , ,

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NBP2-36463AF350
Alexa Fluor 405	NBP2-36463AF405
Alexa Fluor 488	NBP2-36463AF488
Alexa Fluor 532	NBP2-36463AF532
Alexa Fluor 594	NBP2-36463AF594
Alexa Fluor 647	NBP2-36463AF647
Alexa Fluor 700	NBP2-36463AF700
Alexa Fluor 750	NBP2-36463AF750
Alkaline Phosphatase	NBP1-97162
Biotin	NBP2-36463B
Biotin	NBP1-96846
CoraFluor 1	NBP2-36463CL1
DyLight 350	NBP2-36463UV
DyLight 405	NBP2-36463V
DyLight 488	NBP2-36463G
DyLight 550	NBP2-36463R
DyLight 594	NBP2-36463DL594
DyLight 650	NBP2-36463C
DyLight 680	NBP2-36463FR
DyLight 755	NBP2-36463IR
FITC	NBP1-96877
HRP	NBP2-36463H
HRP	NBP1-96797
Janelia Fluor 525	NBP2-36463JF525
Janelia Fluor 549	NBP2-36463JF549
Janelia Fluor 646	NBP2-36463JF646
Janelia Fluor 669	NBP2-36463JF669
PE/Cy7	NBP2-36463PECY7
Rhodamine	NBP1-96878
Texas Red	NBP1-96845
mFluor Violet 450 SE	NBP2-36463MFV450
mFluor Violet 500 SE	NBP2-36463MFV500
mFluor Violet 610 SE	NBP2-36463MFV610

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At	=	A. thaliana	SyHa	=	Golden Syrian Hamster
All	=	All Species	Ha	=	Hamster
An	=	Animal	Hu	=	Human
ArHa	=	Armenian Hamster	I	=	Insect
Av	=	Avian	Ll	=	Llama
Bb	=	Baboon	Ma	=	Mammal
Ba	=	Bacteria	Mk	=	Monkey
Bv	=	Bovine	Mu	=	Mouse
Ca	=	Canine	Multi	=	Multi-species
Ce	=	C. Elegans	NA	=	Non-species specific
Ch	=	Chicken	Or	=	Orangutan
ChHa	=	Chinese Hamster	Pl	=	Plant
Cm	=	Cynomolgus Monkey	Pm	=	Primate
Do	=	Donkey	Po	=	Porcine
Dr	=	Drosophilia	Rb	=	Rabbit
Ec	=	E. Coli	Rt	=	Rat
Eq	=	Equine	RM	=	Rhesus Macaque
Fe	=	Feline	Sh	=	Sheep
Ft	=	Ferret	Vb	=	Vertebrate
Fi	=	Fish	Vi	=	Virus
Fu	=	Fungi	Xp	=	Xenopus
Ge	=	Gerbil	Ye	=	Yeast
GP	=	Guinea Pig	Ze	=	Zebrafish
Gt	=	Goat

Category

Protein/Target

Applications

Reactivity

Citation

Antibody Validation

Reviews

Host

Available Conjugates

Format

Marker

Modification

Clonality

Clone

Gene Symbol

Images Type

Isotype

Kit Type

Preservative

Protein Or Peptide

Research Areas

Has Sample Size

Promotional Item

Species Glossary

Goat IgG Isotype Control [Unconjugated]

IL-1 beta/IL-1F2 Antibody [Unconjugated]

Rabbit IgG Isotype Control [Unconjugated]

Mouse IgG1 Isotype Control (11711) [Unconjugated]

Rabbit IgG Isotype Control

Rabbit IgG Isotype Control

Human IgG Isotype Control [Unconjugated]

Chicken anti-Goat IgG Secondary Antibody [HRP]

Sheep IgG Isotype Control [Unconjugated]

Rat IgG Isotype Control [Unconjugated]

Goat anti-Rabbit IgG (H+L) Secondary Antibody

Rabbit IgG Isotype Control