RIPK1/RIP1 Antibody (334640) [Unconjugated]

Western blot shows lysates of Raji human Burkitt's lymphoma cell line, Jurkat human acute T cell leukemia cell line, DA3 mouse myeloma cell line, and L6 rat myoblast cell line. PVDF membrane was probed with 0.5 µg/mL of Mouse Anti-Human/Mouse/Rat RIPK1/RIP1 Monoclonal Antibody (Catalog # MAB3585) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). A specific band was detected for RIPK1/RIP1 at approximately 75 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 2.

RIPK1/RIP1 was detected in immersion fixed MCF-7 human breast cancer cell line using Mouse Anti-Human/Mouse/Rat RIPK1/RIP1 Monoclonal Antibody (Catalog # MAB3585) at 25 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Simple Western lane view shows lysates of Jurkat human acute T cell leukemia cell line and MCF‑7 human breast cancer cell line, loaded at 0.2 mg/mL. A specific band was detected for RIPK1/RIP1 at approximately 78 kDa (as indicated) using 10 µg/mL of Mouse Anti-Human/Mouse/Rat RIPK1/RIP1 Monoclonal Antibody (Catalog # MAB3585). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system. Non-specific interaction with the 230 kDa Simple Western standard may be seen with this antibody.

Western blot shows lysates of MCF-7 human breast cancer parental cell line and RIPK1/RIP1 knockout MCF-7 cell line (KO). PVDF membrane was probed with 0.5 µg/mL of Mouse Anti-Human/Mouse/Rat RIPK1/RIP1 Monoclonal Antibody (Catalog # MAB3585) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for RIPK1/RIP1 at approximately 75 kDa (as indicated) in the parental MCF-7 cell line, but is not detectable in knockout MCF-7 cell line. GAPDH (Catalog # MAB5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Proteomic analysis identified proteins upregulated in the presence of pro-survival HSP70/BAG1S complex. a U2OS MYC-ER cells expressing ectopic vector, BAG1S, or BAG1 delta S depleted of endogenous BAG1 protein. MYC activity induced for 12 or 24 h with ±100 nM 4-OHT treatment. Lysates analyzed via IB to detect changes in known HSP70 chaperone client proteins GCR, XIAP and RAF1. b Schematic of experimental conditions representing endogenous BAG1 (vector - shLUC), BAG1 knockdown (vector - shBAG1), BAG1S only (BAG1S - shBAG1), or BAG1 delta S only (BAG1 delta S - shBAG1) evaluated for differences in global protein levels. Proteomics analysis outlined with exclusion criteria for significant protein differences between samples. c Efficient knockdown of endogenous BAG1 and rescue of BAG1S and BAG1 delta S shown by IB for samples subjected to proteomics analysis. d Proteomic hits assessed based on schematic of compiled proteins with ≥|1.5| fold change in knockdown compared to control and p ≤ 0.05 across all conditions. Protein expression levels obtained for each sample indexed by specific protein and clustered by UniProt biological process classification. P-values representative of experimental triplicates submitted for proteomic assessment. e Venn diagram showing proteins partially rescued with reintroduction of BAG1S or BAG1 delta S. Increase of ≥10% constitutes a partial rescue. Overlapping proteins with BAG1S or BAG1 delta S indicative of proteins rescued by either ectopic protein. f Verification of proteomics via detection of BAG1S rescued targets SLC7A6 and POLR1D by IB. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/30902071), licensed under a CC-BY license. Not internally tested by R&D Systems.

RPE flatmounts are viable for at least one week after culture in vitro. (A) RPE flatmounts were prepared from 2- and 9-month-old wild-type C57BL/6J mice and cultured in complete medium with or without H2O2 for up to one week. Protein lysates were prepared from the RPE flatmounts and subjected to Western blotting analysis. The cell death marker RIP1 was used to evaluate cell viability; vinculin was used as an internal control. As a positive control: RPE flatmounts treated with either 1 mM or 10 mM H2O2 had significant expression of RIP1. Further, the RPE flatmount that was cultured for 7 days without changing the medium (mishandled sample) showed a slight amount of RIP1, whereas the RPE flatmounts cultured with frequent medium changes did not express RIP1, suggesting that the RPE cells obtained even from older mice (9 months) were viable after at least 7 days of culture. (B) RPE flatmounts were obtained from 3-month-old wild-type C57BL/6J mice and cultured in complete medium with or without H2O2 treatment for up to one week. RPE flatmounts were then fixed in 2.5% glutaraldehyde and processed for transmission electron microscopy (TEM). The TEM imaging of the RPE flatmounts cultured for 16 h, 3 days, and 7 days showed that these RPE cells have normal nuclei (yellow arrows) and organelles, such as mitochondria (yellow arrowheads), and abundant microvilli. H2O2-treated RPE flatmounts showed cell death symptoms, including chromatin condensation and fragmentation (red arrow), a disrupted cell membrane, complete loss of microvilli, a translucent cytoplasm, and severely damaged mitochondria (red arrowheads). (C) ZO-1 immunostaining on 2-month-old RPE flatmounts cultured for 3 days showed robust ZO-1 expression, indicating RPE cell integrity. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/34769409), licensed under a CC-BY license. Not internally tested by R&D Systems.

• Reactivity: Hu, Mu, Rt
• Applications: WB, Simple Western, ICC/IF, KO
• Clone: 334640
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated
• Concentration: LYOPH

RIPK1/RIP1 Antibody (334640) [Unconjugated] Summary

• Immunogen: E. coli-derived recombinant human RIPK1/RIP1
  Met1-Asn671
  Accession # Q13546
• Specificity: Detects human, mouse and rat RIPK1/RIP1 in Western blots.
• Source: N/A
• Isotype: IgG1
• Clonality: Monoclonal
• Host: Mouse
• Gene: RIPK1
• Purity Statement: Protein A or G purified from hybridoma culture supernatant

Applications/Dilutions

• Dilutions:
  • Immunocytochemistry 8-25 ug/mL
  • Knockout Validated
  • Simple Western 10 ug/mL
  • Western Blot 0.5 ug/mL
• Application Notes: In Simple Western only 10-15 uL of the recommended dilution is used per data point.

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.5 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for RIPK1/RIP1 Antibody (334640) [Unconjugated]

• Cell death protein RIP
• EC 2.7.11.1
• receptor (TNFRSF)-interacting serine-threonine kinase 1
• receptor interacting protein
• Receptor-interacting protein 1
• receptor-interacting serine/threonine-protein kinase 1
• RIP
• Rip1 kinase
• RIP1
• RIP-1
• RIPFLJ39204
• RIPK1
• Serine/threonine-protein kinase RIP

Background

Receptor-Interacting Protein 1 (RIP1, also known as RIPK1) is a 671 amino acid (aa) 75 kDa protein that contains an N-terminal protein kinase domain, a C-terminal death domain, and a unique internal region called the intermediate domain. RIP1 is a serine/threonine protein kinase and is constitutively expressed in many tissues. RIP1 interacts with the cytoplasmic death domain of FAS and TNF receptors and is an important element in the signal transduction machinery that mediates apoptosis. RIP1 has been shown to interact with a number of proteins including TRADD, TRAF1, TRAF2, and TRAF3, to form larger signaling complexes. These complexes, in turn, activate specific signaling cascades, such as NF kappa B. RIP1 also interacts through the C-terminal RIP homotypic interaction motif (RHIM) of TRIF in TLR3 dependent activation of NF kappa B.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for RIPK1/RIP1 Antibody (MAB3585)(17)

Publications using MAB3585

• Ai-Ping Tong, Liang-Xue Zhou, Hao-Xiang Wang, Long Zhao, Hao-Xiang Wang, Kun-Hong Zhong et al. Necrostatin-1 decreases necroptosis and inflammatory markers after intraventricular hemorrhage in mice Neural Regeneration Research 2022-06-06 [PMID: 35662218] (Western Blot, Mouse)
• Ai-Ping Tong, Liang-Xue Zhou, Hao-Xiang Wang, Long Zhao, Hao-Xiang Wang, Kun-Hong Zhong et al. Necrostatin-1 decreases necroptosis and inflammatory markers after intraventricular hemorrhage in mice Neural Regeneration Research 6/6/2022 [PMID: 35662218]
• F Yu, W Tian, J Dong Anagliptin prevents lipopolysaccharide (LPS)- induced inflammation and activation of macrophages International immunopharmacology, 2022-01-16;104(0):108514. 2022-01-16 [PMID: 35045357] (Western Blot, Mouse)
• P Shang, NA Stepicheva, H Liu, O Chowdhury, J Franks, M Sun, S Hose, S Ghosh, M Yazdankhah, A Strizhakov, DB Stolz, JS Zigler, D Sinha A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for In Vitro Studies in AMD International Journal of Molecular Sciences, 2021-11-05;22(21):. 2021-11-05 [PMID: 34769409] (Western Blot, Mouse)
• Peng Shang, Nadezda Stepicheva, Kenneth Teel, Austin McCauley, Christopher Scott Fitting, Stacey Hose et al. beta A3/A1-crystallin regulates apical polarity and EGFR endocytosis in retinal pigmented epithelial cells Communications Biology 2021-07-08 [PMID: 34239035] (Western Blot, Mouse)
• R Cheng, X Liu, Z Wang, K Tang ABT?737, a Bcl?2 family inhibitor, has a synergistic effect with apoptosis by inducing urothelial carcinoma cell necroptosis Molecular Medicine Reports, 2021-03-31;23(6):. 2021-03-31 [PMID: 33786632] (Western Blot, Human)
• R Baidya, J Gautheron, DHG Crawford, H Wang, KR Bridle Inhibition of MLKL Attenuates Necroptotic Cell Death in a Murine Cell Model of Ischaemia Injury Journal of Clinical Medicine, 2021-01-08;10(2):. 2021-01-08 [PMID: 33435617] (Flow Cytometry, Western Blot, Mouse)
• Sun C, Chesnokov V, Larson G, Itakura K Glucosamine Enhances TRAIL-Induced Apoptosis in the Prostate Cancer Cell Line DU145 Medicines (Basel) 10/15/2019 [PMID: 31618900]
• VJ Gennaro, H Wedegaertn, SB McMahon Interaction between the BAG1S isoform and HSP70 mediates the stability of anti-apoptotic proteins and the survival of osteosarcoma cells expressing oncogenic MYC BMC Cancer, 2019-03-22;19(1):258. 2019-03-22 [PMID: 30902071] (Western Blot, Human)
• NF Luz, R Khouri, J Van Weyenb, DL Zanette, PP Fiuza, A Noronha, A Barral, VS Boaventura, DB Prates, FK Chan, BB Andrade, VM Borges Leishmania braziliensis Subverts Necroptosis by Modulating RIPK3 Expression Front Microbiol, 2018-09-28;9(0):2283. 2018-09-28 [PMID: 30323793] (Western Blot, Human)
• J Chen, S Wang, R Fu, M Zhou, T Zhang, W Pan, N Yang, Y Huang RIP3 dependent NLRP3 inflammasome activation is implicated in acute lung injury in mice J Transl Med, 2018-08-20;16(1):233. 2018-08-20 [PMID: 30126430] (Western Blot, Human)
• R Chen, J Xu, Y She, T Jiang, S Zhou, H Shi, C Li Necrostatin-1 protects C2C12 myotubes from CoCl2-induced hypoxia Int. J. Mol. Med., 2018-02-06;41(5):2565-2572. 2018-02-06 [PMID: 29436688] (Western Blot, Mouse)
• K Ito, Y Eguchi, Y Imagawa, S Akai, H Mochizuki, Y Tsujimoto MPP+ induces necrostatin-1- and ferrostatin-1-sensitive necrotic death of neuronal SH-SY5Y cells Cell Death Discov, 2017-02-27;3(0):17013. 2017-02-27 [PMID: 28250973] (Western Blot, Human)
• Hypoxia potentiates the cytotoxic effect of piperlongumine in pheochromocytoma models Oncotarget, 2016-06-28;7(26):40531-40545. 2016-06-28 [PMID: 27244895] (Western Blot, Mouse)
• R Oshima, T Hasegawa, K Tamai, N Sugeno, S Yoshida, J Kobayashi, A Kikuchi, T Baba, A Futatsugi, I Sato, K Satoh, A Takeda, M Aoki, N Tanaka ESCRT-0 dysfunction compromises autophagic degradation of protein aggregates and facilitates ER stress-mediated neurodegeneration via apoptotic and necroptotic pathways Sci Rep, 2016-04-26;6(0):24997. 2016-04-26 [PMID: 27112194] (Western Blot, Rat)
• Xu J, Wu F, Tian D, Wang J, Zheng Z, Xia N Open reading frame 3 of genotype 1 hepatitis E virus inhibits nuclear factor-kappaappa B signaling induced by tumor necrosis factor-alpha in human A549 lung epithelial cells. PLoS ONE, 2014-06-24;9(6):e100787. 2014-06-24 [PMID: 24959724] (Western Blot, Human)
• Ikner A, Ashkenazi A TWEAK Induces Apoptosis through a Death-signaling Complex Comprising Receptor-interacting Protein 1 (RIP1), Fas-associated Death Domain (FADD), and Caspase-8. J. Biol. Chem., 2011-04-27;286(24):21546-54. 2011-04-27 [PMID: 21525013] (Western Blot, Human)

Bioinformatics

• Gene Symbol: RIPK1
• Uniprot:
  • Q13546()