Product Search for e. coli: Novus Biologicals

NB200-579

E. Coli Antibody - BSA Free

(9 Publications)

0.5 ml

Reactivity:

Mu, All-NA

Species Glossary

Reactivity: Mu, All-NA

Applications:

, ,

Applications: , ,

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Conjugate:

Unconjugated

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NB100-62526

E. Coli Antibody - BSA Free

(1 Publication)

0.5 ml

Reactivity:

Ba

Species Glossary

Reactivity: Ba

Applications:

Applications:

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Conjugate	Catalog #	Availability	Size	Price
Biotin	NB100-62528-500ul
FITC	NB100-64448-500ul

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB100-62528-500ul
BSA Free	NB100-62526-0.1ml

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NB100-64448

E. Coli Antibody [FITC]

(2 Publications)

500 ul

Reactivity:

Ba

Species Glossary

Reactivity: Ba

Applications:

Applications:

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Conjugate:

FITC

Available Conjugates:

Conjugate	Catalog #	Availability	Size	Price
Biotin	NB100-62528-500ul

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB100-62526
BSA Free	NB100-62526-0.1ml

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NB100-62552

E. Coli Antibody [Biotin]

500 ul

Reactivity:

Ba

Species Glossary

Reactivity: Ba

Applications:

Applications:

Host:

Goat Polyclonal

Host: Goat Polyclonal

Conjugate:

Biotin

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NBP2-53146

E. Coli O157 Antibody (9.88)

(1 Publication)

100 μg

Reactivity:

Ec

Species Glossary

Reactivity: Ec

Applications:

,

Applications: ,

Host:

Mouse Monoclonal

Host: Mouse Monoclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NBP2-54353AF350
Alexa Fluor 405	NBP2-54353AF405
Alexa Fluor 488	NBP2-54353AF488
Alexa Fluor 532	NBP2-54353AF532
Alexa Fluor 594	NBP2-54353AF594
Alexa Fluor 647	NBP2-54353AF647
Alexa Fluor 700	NBP2-54353AF700
Alexa Fluor 750	NBP2-54353AF750
Biotin	NBP2-54353B-0.1ml
DyLight 350	NBP2-54353UV-0.1ml
DyLight 405	NBP2-54353V-0.1ml
DyLight 488	NBP2-54353G
DyLight 550	NBP2-54353R-0.1ml
DyLight 594	NBP2-54353DL594
DyLight 650	NBP2-54353C-0.1ml
DyLight 680	NBP2-54353FR-0.1ml
DyLight 755	NBP2-54353IR-0.1ml
FITC	NBP2-54353F-0.1ml
HRP	NBP2-54353H
Janelia Fluor 549	NBP2-54353JF549
Janelia Fluor 646	NBP2-54353JF646

Available Formulations

Formulation	Catalog #	Availability	Price
Azide and BSA Free	NBP2-54353-100ug

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NB100-64541

E. Coli 015 Antibody (15/443) - BSA Free

0.1 mg

Reactivity:

Ba

Species Glossary

Reactivity: Ba

Applications:

Applications:

Host:

Mouse Monoclonal

Host: Mouse Monoclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB100-64541AF350
Alexa Fluor 532	NB100-64541AF532
Alexa Fluor 594	NB100-64541AF594
Alexa Fluor 750	NB100-64541AF750
Biotin	NB100-64541B
DyLight 550	NB100-64541R
DyLight 594	NB100-64541DL594
DyLight 650	NB100-64541C
FITC	NB100-64541F
HRP	NB100-64541H
Janelia Fluor 549	NB100-64541JF549
Janelia Fluor 646	NB100-64541JF646

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB100-64541-0.025mg

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NB100-64543

E. Coli J5 LPS Antibody (GNE13-337.5 (2D7/1)) - BSA Free

(1 Publication)

0.1 mg

Reactivity:

Ba

Species Glossary

Reactivity: Ba

Applications:

,

Applications: ,

Host:

Mouse Monoclonal

Host: Mouse Monoclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB100-64543AF350
Alexa Fluor 405	NB100-64543AF405
Alexa Fluor 488	NB100-64543AF488
Alexa Fluor 532	NB100-64543AF532
Alexa Fluor 594	NB100-64543AF594
Alexa Fluor 647	NB100-64543AF647
Alexa Fluor 700	NB100-64543AF700
Alexa Fluor 750	NB100-64543AF750
DyLight 350	NB100-64543UV
DyLight 405	NB100-64543V
DyLight 488	NB100-64543G
DyLight 550	NB100-64543R
DyLight 594	NB100-64543DL594
DyLight 650	NB100-64543C
DyLight 680	NB100-64543FR
DyLight 755	NB100-64543IR

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB100-64543-0.025mg

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NB100-62551

E. Coli J5 LPS Antibody - BSA Free

0.5 ml

Reactivity:

Ba

Species Glossary

Reactivity: Ba

Applications:

Applications:

Host:

Sheep Polyclonal

Host: Sheep Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB100-62551-0.1ml

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NB100-64543IR

E. Coli J5 LPS Antibody (2D7/1) [DyLight 755]

0.1 ml

Reactivity:

Ba

Species Glossary

Reactivity: Ba

Host:

Mouse Monoclonal

Host: Mouse Monoclonal

Conjugate:

DyLight 755

Available Conjugates:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB100-64543AF350
Alexa Fluor 405	NB100-64543AF405
Alexa Fluor 488	NB100-64543AF488
Alexa Fluor 532	NB100-64543AF532
Alexa Fluor 594	NB100-64543AF594
Alexa Fluor 647	NB100-64543AF647
Alexa Fluor 700	NB100-64543AF700
Alexa Fluor 750	NB100-64543AF750
DyLight 350	NB100-64543UV
DyLight 405	NB100-64543V
DyLight 488	NB100-64543G
DyLight 550	NB100-64543R
DyLight 594	NB100-64543DL594
DyLight 650	NB100-64543C
DyLight 680	NB100-64543FR

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB100-64543
BSA Free	NB100-64543-0.025mg

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AF-401-NA

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

(3 Reviews)

(570 Publications)

100 μg

AF-401-NA

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.

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

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; CTS008) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Frozen Tissue Sections.

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 (Catalog # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Recombinant MouseIL‑1 beta /IL‑1F2 (Catalog # 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 (Catalog # 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.

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 (Catalog # VC006). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (Catalog # 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.

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 p>0.001,***p<0.001, ns = not significant ) as determined by one way ANOVA with Tukey's post test. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0040722), licensed under a CC-BY license. Not internally tested by R&D Systems.

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 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.

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.

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.

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.

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

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, along with 200ng of plasmids encoding mCASP-1 and mIL-1 beta , and 200ng empty vector to normalize plasmid quantities. Lower quantities of NLRP1B were transfected as compared to panel D so to avoid spontaneous NLPR1B activation. For panels B-E, 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.

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.

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.

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.

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.

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.

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.

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.

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.

Reactivity:

Mu

Species Glossary

Reactivity: Mu

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, , , ,

Applications: , , , ,

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Goat Polyclonal

Host: Goat Polyclonal

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Sample Size Available

Sizes: Sample Size Available

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Unconjugated

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Conjugate	Catalog #	Availability	Size	Price
Biotin	BAF401

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Carrier Free	AF-401-SP

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NBP2-25295

LPS from E. Coli, TLR4 ligand

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(10 Publications)

1 mg

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IMG-2204

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AF1924

SOX17 Antibody [Unconjugated]

(3 Reviews)

(509 Publications)

100 μg

AF1924

Western blot shows lysates of BG01V human embryonic stem cells untreated (-) or endoderm differentiated (+). PVDF membrane was probed with 1 µg/mL of Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF109). A specific band was detected for SOX17 at approximately 55 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Endoderm-differentiated D3 mouse embryonic stem cell line was fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. SOX17/DNA complexes were immunoprecipitated using 5 µg Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) or control antibody (AB-108-C) for 15 minutes in an ultrasonic bath, followed by Biotinylated Anti-Goat IgG Secondary Antibody (BAF109). Immunocomplexes were captured using 50 µL of MagCellect Streptavidin Ferrofluid (MAG999) and DNA was purified using chelating resin solution. Thep21promoter was detected by standard PCR.

SOX17 was detected in immersion fixed B16 mouse melanoma cell line using 10 µg/mL Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) for 3 hours at room temperature. Cells were stained with the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; NL001) and counter-stained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

SOX17 was detected in immersion fixed endoderm differentiated BG01V human embryonic stem cells using 10 µg/mL Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) for 3 hours at room temperature. Cells were stained with the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red, upper panel; NL001) and counterstained with DAPI (blue, lower panel). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Simple Western lane view shows lysates of BG01V human embryonic stem cells untreated (-) or endoderm differentiated (+), loaded at 0.2 mg/mL. A specific band was detected for SOX17 at approximately 59 kDa (as indicated) using 10 µg/mL of Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Simple Western lane view shows lysates of iBJ6 human induced pluripotent stem cell line untreated (-) or endoderm differentiated (+), loaded at 0.2 mg/mL. A specific band was detected for SOX17 at approximately 58 kDa (as indicated) using 10 µg/mL of Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Western blot shows lysates of SK‑OV‑3 human ovarian adenocarcinoma cell line, OVCAR‑3 human ovarian carcinoma cell line. PVDF membrane was probed with 2 µg/mL of Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for SOX17 at approximately 60 kDa (as indicated). This experiment was conducted under reducing conditions and using Western Blot Buffer Group 1.

Generation and characterization of systemic lupus erythematosus (SLE)‐specific human‐induced pluripotent stem cells (hiPSCs). Immunostaining of Otx2 (ectoderm marker), Sox17 (endoderm marker), Brachyury (mesoderm marker) and co‐staining with DAPI (blue) in embryoid bodies (EBs) derived from hiPSCs‐SLE. Scale bar, 50 μm. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31536674), licensed under a CC-BY licence.

Concordant expression of developmental programs across organoids from human iPSC lines. IF staining of D29 kidney organoid (AS line, ML protocol) for SOX17 and CD31, markers of endothelial cells. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31784515), licensed under a CC-BY licence.

Activin-A levels during Day 0–1 modulate cardiomyocyte (CM) vs. definitive endoderm (DE) differentiation. Pluripotent H1 ESCs were induced by changing medium to RPMI/B27 (no insulin), including CHIR (7.5 μmol/L) during Day 0–1 and IWP (5 μmol/L) during Days 3–5. Activin-A was included at the indicated levels during Day 0–1. Insulin (4,000 ng/ml) was included after Day 7. a-e shows cells double-immunostained on Day 5 for Oct4 (red) and Sox17 (green); e is a positive control wherein cells were induced to DE with Activin-A (50 ng/ml) and Bmp4 (10 ng/ml) during Days 0–5. f-i shows cells immunostained with MF20 mAb on Day 14 to detect cardiomyocytes. Scale bar: 200 μm. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0118670), licensed under a CC-BY licence.

In vitro and in-TiPSCs. Immunofluorescence staining for Sox17 (endodermal marker), alpha SMA (mesodermal marker), and Nestin (ectodermal marker) in each human TiPSCs1-derived differentiated cell in vitro. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0097397), licensed under a CC-BY licence.

Differentiation of hESCs into definitive endoderm cells by Activin A with CHIR99021. Immunofluorescence analysis of the expression of SOX17 and FoxA2 for Activin A with 2 µM CHIR99021-induced differentiated cells on day 3. Scale bar, 100 µm. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31601782), licensed under a CC-BY licence.

HIF-1 alpha activates transcription of Sox17. Western blot analysis in control human lung microvascular endothelial cells (HLMVECs) and HLMVECs for which CRISPR/Cas9 was used to delete HIFs. ECs were treated with the HIF prolyl hydroxylase inhibitor DMOG to induce HIF expression. DMOG (1 mM) increased HIF-1 alpha and HIF-2 alpha protein expression in control ECs but not in ECs lacking HIF-1 alpha or HIF-2 alpha . Induction of HIF expression was coupled to Sox17 upregulation. n = 3. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY licence.

Features of ESC-INTPSCs intermediate between ESCs and EpiSCs. Expression of OCT3/4 and SOX17. Scale bar, 100 µm. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0095329), licensed under a CC-BY licence.

p38-Mapk14/11 inhibition during murine blastocyst maturation blocks primitive endoderm (PrE) differentiation/maturation. Representative single confocal z-plane micrographs of vehicle control-treated (+DMSO) or p38-Mapk14/11 inhibited (+SB220025) late-blastocyst stage/equivalent embryos, immunofluorescently stained for indicated ICM cell lineage markers (Nanog in green and Gata4, Sox17 and Gata6 in red, plus DAPI DNA stain in blue). Examples of cells classified as TE, PrE and EPI are marked with an asterisk, arrowhead and arrow, respectively. Scale bar, 15 µm. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27605380), licensed under a CC-BY licence.

Endothelial cell (EC) specific deletion of Sox17 (Sox17EC−/−) in mice prevents endothelial regeneration. Western blot analysis of Sox17 protein expression in isolated ECs obtained from flushed lungs of Sox17EC−/− and control mice. n = 4. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY licence.

Characterization of hybrid cells.(A) Both ES-pNSC and pES-NSC hybrid cells are positive for alkaline phosphatase staining (100 ×). (B) RT-PCR analysis of Oct4, Nanog, Sox2, and Nestin expression in fusion partner and reprogrammed hybrid cells. Pluripotency markers, Oct4 and Nanog, which were not expressed in NSCs and pNSCs were expressed in GFP+ fusion hybrid cells. On the other hand, Nestin, which was expressed in NSCs and pNSCs was silenced after forming GFP+ fusion hybrid cells. (C) Immunocytochemistry analysis of Oct4 and Nanog in ES-pNSC and pES-NSC hybrid cells (100 ×). (D)In vitro differentiation of ES-pNSC and pES-NSC hybrid cells into ectoderm (Tuj1), mesoderm (SMA), and endoderm (Sox17) lineages (200 ×). (E) In vivo differentiation potential of ES-pNSC and pES-NSC hybrid cells through teratoma assay. These hybrid cells were contributed to secretory epithelium (ectoderm), cartilage (mesoderm) and gut epithelium (endoderm), which were stained with PAS, Asian blue, and hematoxylin eosin, respectively. Each tissue was indicated by arrow head. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0156491), licensed under a CC-BY license. Not internally tested by R&D Systems.

In vitro and in-TiPSCs.(A): Immunofluorescence staining for Sox17 (endodermal marker), alpha SMA (mesodermal marker), and Nestin (ectodermal marker) in each TiPSCs1-derived differentiated cell in vitro. (B): Gross morphology of representative teratomas derived from TiPSCs1 in vivo (hematoxylin and eosin staining). Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0097397), licensed under a CC-BY license. Not internally tested by R&D Systems.

Activation of Sox17 at onset of EC regeneration and Sox17-mediated Cyclin E1 expression. a qPCR analysis of gene expression in sorted CD31+ cells from mTmG-Scl mice before and after injury (12 mg/kg i.p.). Sox17, Vegfr2, and Ccne1 increased significantly at day 2 post-LPS compared to baseline. n = 3. Color scale: the fold change increases from red to white to green color. b Western blot analysis in fresh isolated ECs from wild-type mice and quantification c showed a 5-fold increase in Sox17 protein expression within 1 day following injury compared to baseline and followed by recovery within 3 days post-LPS. n = 3. d, e Western blot analysis of cultured HLMVECs in which Sox17 was overexpressed showed 2.5x fold increase in Cyclin E1 protein expression relative to control cells. n = 3. OE, overexpression. f Representation of the CCNE1 promoter region with Sox17 binding sites (circled numbers) and their sequences. g HLMVECs were retrovirally transduced with Sox17 or control plasmid for 3 days, and Ch-IP assay followed by qPCR was performed to amplify Sox17 binding sites in the CCNE1 promoter. n = 3. h 293T cells were transfected with a Sox17 overexpression plasmid containing CCNE1 luciferase reporter constructs. Luciferase values were normalized to Renilla luciferase control reporter values. A schematic representation of corresponding deletion constructs is presented in the right panel. n = 3 and duplicates per sample. **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using one-way ANOVA for (c) and two-way ANOVA with Bonferroni post-tests for (e, g, h) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY license. Not internally tested by R&D Systems.

Generation and characterization of systemic lupus erythematosus (SLE)‐specific human‐induced pluripotent stem cells (hiPSCs). (A) Dermal fibroblasts derived from patient with SLE were reprogrammed into iPSCs using Sendai virus vectors and three clones (#1, #2 and #3) were characterized. RT‐PCR confirms the loss of transgenes in hiPSCs‐SLE (lanes 1, 2 and 3), presence (lane 4) in infected fibroblasts (ipF‐SLE) and absence of Sendai viral transgenes in parental fibroblasts (pF‐SLE) (lane 5). Full‐length gels are presented in File S2. (B) The hiPSCs‐SLE colonies expressed alkaline phosphatase. Scale bar, 500 μm. (C) RT‐qPCR analysis of pluripotency genes OCT4, NANOG, SOX2 and REX1 was performed in fibroblasts and in hiPSCs derived from patient with SLE. All expression values are normalized to GAPDH and relative donor fibroblasts. Data are mean ± SEM and all statistical analysis was made between hiPSCs‐SLE clones and relative fibroblasts by Student's t test showing P‐values ≤ .05 in each comparison. (D) PluriTest assays combines novelty score (blue) on x‐axis and pluripotency score (red) on y‐axis. hiPSCs‐SLE localize in the red cloud suggesting the empirical distribution of pluripotent cells compared to non‐pluripotent blue cloud. (E) Representative images of M‐FISH staining show normal karyotypes of hiPSCs‐SLE clones. (F) Immunofluorescence analysis of pluripotent stem cell markers Nanog (green), Oct4 (red) and co‐staining with DAPI (blue) in hiPSCs‐SLE. Scale bar, 50 μm. (G) Representative images of floating and adherent EBs derived from hiPSCs‐SLE at differentiation day 8 and 18, respectively. Scale bar, 500 μm. (H) RT‐qPCR results confirm the capability of hiPSCs‐SLE to differentiate into all three germ layers. The expression levels of GATA4, HAND1 and PAX6 in EBs are relative to undifferentiated hiPSCs. All expression values are normalized to GAPDH and relative hiPSCs. Data are mean ± SEM and all statistical analysis was made between EBs‐SLE and relative hiPSCs‐SLE clones by Student's t test showing P‐values ≤ .05 in each comparison. (I) Immunostaining of Otx2 (ectoderm marker), Sox17 (endoderm marker), Brachyury (mesoderm marker) and co‐staining with DAPI (blue) in EBs derived from hiPSCs‐SLE. Scale bar, 50 μm Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31536674), licensed under a CC-BY license. Not internally tested by R&D Systems.

soHIF-1 alpha signaling induces Sox17 expression. a MPO activity of flushed lung sample from mice challenged with LPS (12 mg/kg i.p.) for 6 and 24 h. n = 3. b Western blot analysis of wild-type mice lung before and after LPS-induced injury (12 mg/kg i.p.) and its quantification c showed that HIF-1 alpha protein expression increased within 6 h post-LPS and remained increased until day 2. n = 3. d Western blot analysis in freshly isolated ECs from Hif1afl/fl mice and quantification e showed significantly increased Sox17 protein expression after injury when compared to baseline levels. n = 3. f Western blot analysis in freshly isolated ECs from Hif1aEC−/− mice and quantification g showed no significant difference in Sox17 protein expression before and after injury. n = 3. **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using one-way ANOVA for (b, c, e, g). Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY license. Not internally tested by R&D Systems.

PGCLCs derived from hc-iPSCs.Immunofluorescent staining of (A) BLIMP1 (green color) and (B) OCT4 (green color) in 4-day-old EBs cultured with PGC induction medium on hc-iPSC-1, -2, -5 and -6 (third to fifth column), PBMC derived iPSC0102 and iPSC0207 (right two column). H9 hESCs (left column) adapted in 4i medium was also detected for BLIMP1 and OCT4 antibodies. Noted that there was no BLIMP1 signals detected in undifferentiated H9 in 4i condition. All samples were doubly stained with SOX17 antibody (red color) sequentially. DAPI was used for counterstain (blue). Scale bar = 50μm. (C) Quantification of immunofluoresent staining of PGCLCs. BLIMP1/SOX17 and OCT4/SOX17 double positive cells were counted for PGCLCs and normalized with the number of DAPI. Percentage of PGCLCs per EB of all lines was presented and significance was compared with control hESC H9. ** indicates P<0.005. *** indicates P<0.001. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0165715), licensed under a CC-BY license. Not internally tested by R&D Systems.

Activin-A levels during Day 0–1 modulate CM vs. DE differentiation.Pluripotent H1 ESCs were induced by changing medium to RPMI/B27 (no insulin), including CHIR (7.5 μmol/L) during Day 0–1 and IWP (5 μmol/L) during Days 3–5. Activin-A was included at the indicated levels during Day 0–1. Insulin (4,000 ng/ml) was included after Day 7. Panel A, a-e shows cells double-immunostained on Day 5 for Oct4 (red) and Sox17 (green); e is a positive control wherein cells were induced to DE with Activin-A (50 ng/ml) and Bmp4 (10 ng/ml) during Days 0–5. Panel A f-i shows cells immunostained with MF20 mAb on Day 14 to detect cardiomyocytes. Panel B depicts the effect of Activin-A levels during Day 0–1 on cardiomyocyte differentiation at Day 14, determined by flow cytometry using anti-cTnT. Cultures treated with 10 ng/ml Activin-A began to rhythmically contract at Day 6. Cells treated with 50 or 100 ng/ml Activin-A did not beat at any time. Bars indicate the average values combined from multiple experiments. Vertical lines = ±SEM. P-values were calculated by Student’s t-test. The p-value over the bar denoting 10 ng/ml Activin-A is relative to cells treated with CHIR only (0 ng/ml Activin-A), whereas the p-values over the bars denoting 50 and 100 ng/ml Activin-A are relative to cells treated with 10 ng/ml Activin-A. The size bar in Aa, which pertains to panels a-i, = 200 μm. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0118670), licensed under a CC-BY license. Not internally tested by R&D Systems.

Features of ESC-INTPSCs intermediate between ESCs and EpiSCs.(A) Comparison of gene expression in ESCs, EpiSCs, and ESC-INTPSCs. (B) The number of AP-positive colonies formed in the LIF/2i condition four days after 1000 cells were seeded per dish. (C) Proliferation of ESCs, EpiSCs, and ESC-INTPSCs with or without JAKi. (D) Expression of ESRRB and FOXA2. White arrows indicate ESRRB- and FOXA2-double–positive cells. Scale bar, 100 µm. (E) Percentage of ESRRB-positive or ESRRB- and FOXA2-double–positive cells in ESCs, ESC-INTPSCs, and EpiSCs. (F) Expression of OCT3/4 and SOX17. Scale bar, 100 µm. (G) The number of Oct3/4-GFP–positive or –negative colonies derived from single cells at day 4 and 8. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0095329), licensed under a CC-BY license. Not internally tested by R&D Systems.

EC specific deletion of Sox17 (Sox17EC−/−) in mice prevents endothelial regeneration. a Schematic diagram of Sox17fl/fl mice crossed with Scl-CreERT2 to delete Sox17 in ECs. After tamoxifen feeding for five days and rest for another four weeks, mice are challenged with LPS (sub-lethal 8 mg/kg, i.p.) for analysis. b Western blot analysis of Sox17 protein expression in isolated ECs obtained from flushed lungs of Sox17EC−/− and control mice. n = 4. c Quantification of b shows 80% deletion of Sox17 in ECs of Sox17EC−/− mice compared to control mice. n = 4. d Time course of lung transvascular permeability following LPS challenge in Sox17EC−/− and Sox17fl/fl mice. n = 4. While control mouse lungs showed increased endothelial permeability at day 1 post-LPS and then recovered to baseline by day 5, Sox17EC−/− mice showed prolonged endothelial barrier leakiness post-LPS. e Time course of changes in lung transvascular permeability following LPS challenge was also carried out in Sox17fl/fl mice crossed with Cdh5-CreERT2 mice. n = 4. Similar as in d, these Sox17EC−/− mice also showed persistent leakiness post-LPS while control mice fully recovered. f Survival curve of LPS challenge in Sox17EC−/− and control mice. n = 8 per group. At this sub-lethal dose, all control mice survived whereas half of Sox17EC−/− mice died on day 2 post-LPS with increased mortality on day 3. By day 5, the death rate for control mice is 0 while for Sox17EC−/− mice is 60%. g Flow cytometry analysis of CD31+CD45− ECs among whole lung population in mice following injury. n = 4. In contrast to control mice in which CD31+CD45− EC population gradually recovered with day 3 post-LPS after initial loss of ECs, Sox17EC−/− mice showed significantly delayed restoration of ECs post-LPS period. h Quantification of BrdU+ nuclei in each field of 425 μm2 area in flushed lung cryo-sections from mice following injury. n = 4 mice per group and 6 replicates per sample. Slides are co-stained with CD31-AF488, BrdU-APC, and DAPI. At day 3 post-LPS, the control group showed a significantly higher number of BrdU+ECs compared to baseline. However, Sox17EC−/− mice showed markedly reduced level of BrdU+ECs, indicating reduced EC proliferation. i To assess whether expression of Sox17 in ECs can restore lung endothelial integrity, studies were performed in Sox17EC−/− mice to overexpress Sox17 protein. We used a mixture of 50 μg plasmid (mouse Cdh5 promoter—Flag —Sox17) encapsulated in liposomes, which were injected i.v. 3 h after LPS challenge. j At day 3 post-LPS, liposome vector-treated Sox17EC−/− mice showed marked EC barrier leakiness as assessed by lung transvascular permeability of albumin whereas the Sox17-rescued mice showed markedly reduced endothelial permeability. n = 4. OE, overexpression. *P < 0.05, **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using two-tailed Student’s t-test for (c, j), two-way ANOVA with Bonferroni post-tests for (d, e, g, h) and Log-rank (Mantel-Cox) test for (f) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY license. Not internally tested by R&D Systems.

p38-Mapk14/11 inhibition during blastocyst maturation blocks PrE differentiation/maturation. (a) Experimental schema of p38-Mapk14/11 inhibition (+SB220025), plus vehicle control (+DMSO), and the details of antibodies used to analyse ICM cell lineage marker protein expression by immunofluorescence (IF) in late blastocysts (E4.5); Nanog and Gata4 (+DMSO n = 27, +SB220025 n = 33)—green, Nanog and Sox17 (+DMSO n = 18, +SB220025 n = 20)—red, and Nanog and Gata6 (+DMSO n = 24, +SB220025 n = 27)—blue. (b) Representative single confocal z-plane micrographs of vehicle control-treated (+DMSO) or p38-Mapk14/11 inhibited (+SB220025) late-blastocyst stage/equivalent embryos, immunofluorescently stained for indicated ICM cell lineage markers (Nanog in green and Gata4, Sox17 and Gata6 in red, plus DAPI DNA stain in blue). Examples of cells classified as TE, PrE and EPI are marked with an asterisk, arrowhead and arrow, respectively. Scale bar, 15 µm. (c) Pie charts of the relative cell lineage contribution in vehicle control (+DMSO) and p38-Mapk14/11 inhibited (+SB220025) blastocysts as judged by IF to detect the stated ICM lineage marker proteins. Blue, trophectoderm (TE); yellow, epiblast (EPI—ICM exhibiting exclusive Nanog expression); green, primitive endoderm (PrE—ICM exhibiting exclusive Gata4/6 or Sox17 expression, as appropriate); orange, uncommitted ICM cells (exhibiting co-expression of both Nanog and Gata4/6 or Sox17, as appropriate); and grey, ICM cells negative for either assayed marker. (d) Bar charts show average number of cells allocated to each specified ICM lineage, as judged by the indicated IF staining regime employed. Error bars represent s.e.m. and asterisks denote statistical significant differences in cell number between the vehicle control (+DMSO, black bars) and p38-Mapk14/11 inhibited (+SB220025, grey bars) embryo groups, according to two-tailed Student's t-test, with *p < 0.05 and **p < 0.005 confidence intervals. Yellow asterisk denotes increase in cells positively immunofluorescently staining for both EPI and PrE ICM markers using anti-Nanog and anti-Gata6 (an early PrE marker) antibodies. All individual embryo data used in the preparation of this figure are contained within the electronic supplementary material, table S3. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27605380), licensed under a CC-BY license. Not internally tested by R&D Systems.

Differentiation of hESCs into definitive endoderm cells by Activin A with CHIR99021.a The relative primitive streak (BRACHYRUY, MESP1, and MIXL1) gene expression of the day 1 differentiated cells by adding Activin A (100 ng/mL) with CHIR99021 (2–5 µM) or Wnt3a (25–100 ng/mL) treatments were determined by real-time quantitative PCR (qPCR). None, no WNT signaling pathway activators were used on day 1 for PS differentiation (Activin A only). b, c After 1 day, the medium was changed to Activin A (100 ng/mL) and 1 × CTS-B27 to induce DE differentiation. The definitive endoderm (FOXA2 and SOX17) and mesoderm (MESP2 and HAND1) relative gene expression levels were determined by qPCR. d Immunofluorescence analysis of the expression of SOX17 and FoxA2 for Activin A with 2 µM CHIR99021-induced differentiated cells on day 3. e, f The expression of SOX17 and FoxA2 for Activin A with 2 µM CHIR99021-induced differentiated cells was determined by flow cytometry on day 3. Isotype control antibodies were used as controls. At a specific gene expression, datum points lacking common letters differ, p < 0.05. Data are represented as the mean ± SD. Scale bar, 100 µm Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31601782), licensed under a CC-BY license. Not internally tested by R&D Systems.

HIF-1 alpha activates transcription of Sox17. a Western blot analysis in control HLMVECs and HLMVECs for which CRISPR/Cas9 was used to delete HIFs. ECs were treated with the HIF prolyl hydroxylase inhibitor DMOG to induce HIF expression. DMOG (1 mM) increased HIF-1 alpha and HIF-2 alpha protein expression in control ECs but not in ECs lacking HIF-1 alpha or HIF-2 alpha . Induction of HIF expression was coupled to Sox17 upregulation. n = 3. b Quantification of a showed that protein expression of HIF-1 alpha and HIF-2 alpha in ECs was significantly increased by DMOG treatment. Sox17 showed a 2.5-fold increase in Sox17 expression control DMSO treated ECs. The increase in Sox17 was significantly reduced in HIF-1 alpha -deleted ECs but preserved in HIF-2 alpha -deleted ECs, indicating the importance of HIF-1 alpha in mediating Sox17 expression. n = 3. c Representation of the SOX17 promoter region with 3 HREs indicated by circled numbers, and their respective sequences are displayed. d HLMVECs were exposed to normoxia or 1% O2 (hypoxia) for 8 h. Ch-IP assay followed by qPCR was performed to amplify the HRE regions in the SOX17 promoter. Studies were performed in ECs exposed to either normoxia or hypoxia. n = 3. e 293T cells were transfected with a HIF-1 alpha overexpression plasmid containing SOX17 luciferase reporter constructs. Luciferase values were normalized to Renilla luciferase control reporter values. A schematic representation of corresponding deletion constructs is presented in the right panel. n = 3 and duplicates per sample. Results show that hypoxia activation of SOX17 HRE3 was required for Sox17 expression. **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using two-way ANOVA with Bonferroni post-tests for (b, d, e) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY license. Not internally tested by R&D Systems.

Concordant expression of developmental programs across organoids from four human iPSC lines. a Heatmap of expression patterns for major nephrogenesis markers across organoid differentiation time points (iPSC D0, D7, D15, and D29, averaged across four cell lines, ML protocol) and human adult kidney. Expression values were row-normalized to obtain z-scores; red color indicates positive z-scores. b Canonical (NPHS2) and data-derived (CLDN5) podocyte marker genes superimposed in tSNE plots from D15 organoids (N2 line, ML protocol). c IF staining of D15 kidney organoid (N2 line, ML protocol) for CLDN5 as a marker of early podocyte differentiation derived from the single-cell data. Additional canonical podocyte markers (NPHS1, WT1) and DAPI staining as shown. d IF staining of D29 kidney organoid (AS line, ML protocol) for SOX17 and CD31, markers of endothelial cells. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31784515), 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.

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.

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Reactivity: Hu

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Goat Polyclonal

Host: Goat Polyclonal

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Conjugate	Catalog #	Availability	Size	Price
Allophycocyanin	IC1924A
Biotin	BAF1924
NL557	NL1924R

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Formulation	Catalog #	Availability	Price
Carrier Free	IC1924A
Carrier Free	NL1924R
Carrier Free	AF1924-SP

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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

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E. Coli Antibody - BSA Free

E. Coli Antibody - BSA Free

E. Coli Antibody [FITC]

E. Coli Antibody [Biotin]

E. Coli O157 Antibody (9.88)

E. Coli 015 Antibody (15/443) - BSA Free

E. Coli J5 LPS Antibody (GNE13-337.5 (2D7/1)) - BSA Free

E. Coli J5 LPS Antibody - BSA Free

E. Coli J5 LPS Antibody (2D7/1) [DyLight 755]

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

LPS from E. Coli, TLR4 ligand

SOX17 Antibody [Unconjugated]