Human/Mouse/Rat RIPK1/RIP1 Antibody

Detection of Human/Mouse/Rat RIPK1/RIP1 by Western Blot. 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 in MCF‑7 Human Cell Line. 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.

Detection of Human RIPK1/RIP1 by Simple Western^TM. 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 Human RIPK1/RIP1 Specificity by Using Knockout Cell Line. 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.

Detection of RIPK1/RIP1 by Western Blot 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 (https://pubmed.ncbi.nlm.nih.gov/30902071), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of RIPK1/RIP1 by Western Blot 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 (https://pubmed.ncbi.nlm.nih.gov/34769409), licensed under a CC-BY license. Not internally tested by R&D Systems.