Anti mlkl ab184718

Protein isolation and Western Blot were conducted as previously described^{26 ,27 (link)}. In brief, GCs were lysed after 1 to 5 days of culture using RIPA buffer containing protease and phosphatase inhibitors (PI, Thermo Fisher Scientific, Waltham, USA). A total of 10 µg protein per lane was loaded on a 12 % SDS gel and run under constant current (30 mA/gel). After blotting (100 V, 65 min) and blocking with 5 % non-fat dry milk in Tris-buffered saline with Tween 20 (TBS-T, 50 mM Tris-HCl, 150 mM NaCl, 0.1 % Tween 20, pH 7.4), anti-pRIP1(S166), anti-pRIP3(S227) both from Cell Signaling Technology (Danvers, MA, USA) and anti-pMLKL(T357/S358) antibodies were administered to decorate these phosphorylated proteins. To visualize specific binding, HRP-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Inc., West Grove, PA, USA) was used. As loading controls, anti-RIP1, anti-RIP3 (both from Cell Signaling Technology, Danvers, MA, USA), anti-MLKL (ab184718, Abcam, Cambridge, UK) and anti-β-actin (A5441, Sigma-Aldrich, St. Louis, MI, USA) antibodies were used. Preabsorption of anti-pMLKL was previously published^{27 (link)}. All experiments were carried out five times if not described otherwise.

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).

Poly(I:C) HMW was purchased from InvivoGen (San Diego, California, USA). Smac mimetic (SM-164) was a gift from S. Wang (University of Michigan, Ann Arbor, Michigan, USA). Pan-caspase inhibitor (z-VAD-FMK), GSK’782, necrosulfonamide (NSA), Bay11–7082, U0126, SP600125 and SB203580 were purchased from Calbiochem (Merck Millipore, Darmstadt, Germany). Necrostatin-1 (Nec-1) were purchased from Sigma (St Louis, Missouri, USA). TNF-α was purchased from R&D systems (Minneapolis, Minnesota, USA). TLR3 inhibitor (CuCpt4a) was purchased from APExBIO (Boston, Massachusetts, USA). Antibodies for Western blot were purchased from commercial available providers as following: anti-RIPK1 (610459) was from BD Biosciences (San Jose, California, USA); anti-TLR3 (6961), anti-RIPK3 (8457), anti-cIAP1 (7065), anti-cIAP2 (3130), anti-caspase-8 (9746), anti-caspase-3 (9662), anti-PARP-1 (9542) and anti-actin (4970) were from Cell signaling (Danvers, Massachusetts, USA); anti-MLKL (ab184718) was from Abcam (Cambridge, UK).