Anti ripk1 610459

Anti-RIPK3 (ab72106), anti-MLKL (ab184718), anti-pMLKL (ab187091), and anti-FOXp3 (ab20034) were purchased from Abcam (Cambridge, UK); anti-PD-L1 (E1L3N) was obtained from Cell Signaling Technology (Danvers, Massachusetts, USA); anti-RIPK1 (610459) was bought from BD Biosciences (San Jose, California, USA); anti-CD8 (M7103) was purchased from Dako (Agilent) (Santa Clara, California, USA); and anti-CD163 (NCL-L-CD163) was purchased from Leica Biosystems (Wetzlar, Germany).

Immunoprecipitation and immunoblot analysis were performed as described previously^{35 (link)}. Briefly, the cells were treated as indicated and washed with cold PBS. The cells were harvested with lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1.0% Triton X-100, 1 mM EDTA, and 10% glycerol containing protease inhibitors). Cell extracts were left on ice for 30 min and centrifuged at 13,000 r.p.m for 30 min. Cell lysates were incubated with 2μg of anti-RIPK1 antibody or 2μg of normal mouse IgG antibody (Santa Cruz) overnight at 4°C and then incubated with 15μl of protein A beads for an additional 3h at 4°C. The beads were washed with lysis buffer three times and then boiled in 20μl of 1×SDS loading buffer for 10 min. Samples were loaded on SDS-PAGE gels followed by electroblotting onto PVDF membranes (Millipore). To determine protein levels, immunoblot was performed using the following antibodies: anti-RIPK3 (2283) from Pro Sci; anti-pMLKL (ab196436) from Abcam; anti-RIPK1 (610459) from BD Biosciences; anti-pP38 (9211S), anti-JNK (9252), anti-pERK1/2 (4370), anti-pRIPK1 (S166) (31122S), anti-pJNK (9251) from Cell Signaling, anti-Tim23 (11123-1-AP) from Proteintech, anti-GAPDH (sc-32233) from Santa Cruz, and anti-TRIF (657102) from Biolegend.

Pan-caspase inhibitor, z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone), GSK’782, and necrosulfonamide (NSA) were purchased from Calbiochem (Merck Millipore, Darmstadt, Germany). Gemcitabine and Necrostatin-1 (Nec-1) were from Sigma (St Louis, Missouri, USA). TNF-α was from R&D systems (Minneapolis, Minnesota, USA). Smac mimetic (SM-164) was a gift from S. Wang (University of Michigan, Ann Arbor, Michigan, USA). Antibodies for Western blot were obtained from commercial sources: anti-RIPK1 (610459) and anti-FADD (610400) were from BD Biosciences (San Jose, California, USA); anti-RIPK3 (8457), anti-cIAP1 (7065), anti-cIAP2 (3130), anti-NIK (4994), anti-cyclin D1 (2978), anti-caspase-8 (9746) and anti-actin (4970) were from Cell Signaling (Danvers, Massachusetts, USA); anti-MLKL (ab184718) and anti-phosphorylated MLKL (ab187091) were from Abcam (Cambridge, UK).