Whole cell lysates were prepared with TNE lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% v/v NP-40, 3 mM EDTA, supplemented with Complete protease inhibitor mixture (Roche)). The protein lysates were separated by SDS-PAGE, transferred onto nitrocellulose membranes and reactive proteins were detected with antibodies for RIPK1 (BD Biosciences), RIPK3 (Abnova, Heidelberg, Germany), MLKL or actin (Sigma-Aldrich) via chemiluminescence (Lumiglo, Cell Signaling, Danvers, MA, USA). RIPK3 expression was quantified using the program ImageJ (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). To compare expression levels of RIPK1 and RIPK3 in tumor cell lines, identical amounts of protein (20 μg) were loaded, using lysates from PancTu-I cells as a standard on each gel, and identical exposure times were taken to allow a direct comparison of expression levels. For the quantitative analysis of the relationship between the levels of RIPK3 and the specific sensitivity of the respective tumor cell line to TRAIL/zVAD/CHX-induced programmed necrosis, values for RIPK3 expression were normalized between the gels and calculated relative to CCRF-CEM cells. In all Western blots, detection of actin served as a loading control.
Ripk3
Manufactured by Abnova
Sourced in Germany
RIPK3 is a protein that plays a key role in the regulation of programmed cell death pathways. It is a member of the receptor-interacting protein kinase (RIPK) family and is involved in the initiation of necroptosis, a form of programmed cell death. The RIPK3 protein acts as a central regulator of this process by promoting the phosphorylation and activation of downstream signaling molecules.
Automatically generated - may contain errors
Lab products found in correlation
Complete protease inhibitor mixture, by Roche (1 mentions)
Ripk1, by BD (1 mentions)
Lumiglo, by Cell Signaling Technology (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Ripk3, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Caspase 9, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Mcl 1, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
β actin antibody, by Merck Group (1 mentions)
Cytochrome c, by Cell Signaling Technology (1 mentions)
Flip antibody, by Adipogen (1 mentions)
Ripk1, by Cell Signaling Technology (1 mentions)
2 protocols using ripk3
1
Quantifying RIPK1 and RIPK3 Expression
2
Quantitative Apoptosis Protein Profiling
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The FEBS Journal 288 (2021) 5374-5388 ª 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies obtained from Enzo (Exeter, UK). FLIP antibody was purchased from AdipoGen (San Diego, CA, USA). RIPK1, RIPK3, XIAP, caspase-3, caspase-9, SMAC, Bid, BCL-2, BCL-XL, MCL-1, Bax, Bak APAF-1 and cytochrome c antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). β-actin antibody was purchased from Sigma (St. Louis, MO, USA). Fluorophore-labelled secondary antibodies were acquired from LI-COR Biosciences (Lincoln, NE, USA). Images were captured using an Odyssey Imaging System (LI-COR) at 12-bit dynamic range. Protein expression in reference cell lines (HeLa/ HT29) was quantified against a standard curve of recombinant protein standards or previously quantified and was used to determine absolute quantities in the cell line panel. Recombinant RIPK1, RIPK3, cIAP1, cIAP2 and XIAP were purchased from Abnova (Taoyuan, Taiwan). p-18 caspase-8 was acquired from Sigma, and FLIP and FADD were produced in-house (PGJCCR, Queen's University Belfast) as described in Majkut et al. [17] . Recombinant Bid, BCL-2, BCL-XL, MCL-1, Bax, Bak, APAF-1, procaspase-9, procaspase-3 and SMAC were used to quantify absolute protein concentrations as previously published [16, 18] .
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