α sharpin

The GSK’963 RIPK1 kinase inhibitor was provided by GSK. The following antibodies were used: α-RIPK1 (BD Biosciences, 610459), α-HOIL (gift from Henning Walczak), α-cIAP1 (Enzo, ALX-803-335-C100), α-TNFR1 (Abcam, 19139), α-Actin (Santa Cruz Biotechnology, sc-1615), α-P-p65 (Cell Signaling, 3033), α-p65 (Cell Signaling, 8242), α-IkBα (Santa Cruz, sc-371), α-P-p38 (Cell Signaling, 9215), α-p38 (Cell Signaling, 9212), α-P-JNK (Cell Signaling, 9255), α-JNK (Santa Cruz Biotechnology, sc-571), α-P-ERK (Cell Signaling, 9101), α-ERK (gift from Chris Marshall) α-caspase-8 (Cell Signaling, 9429), α-FLAG [M2] (SIGMA, M8823), α-Ub (Dako, Z0458), α-FLIP (Adipogene, AG-20B-0056), α-FADD (Santa Cruz Biotechnology, sc-6036), α-RIPK3 (ProSci, 2283), α-Tubulin (SIGMA, T9026), α-SHARPIN (Proteintech, 14626-1-AP), α-TRAF2 (Cell Signaling, 4712), α-CD8-PE-Cy7, GR-1-PE-Cy7, CD11c-FITC, CD4-FITC, CD11b-Cy5, B220-FITC (gift from Henning Walczak), α-CD69-PE (eBioscience, 12-0691-82), α-CD3-APC (eBioscience, 47-0032-82), and α-CD16 (eBioscience, 14-0161-82).

DISC lysis buffer (20 mM Tris–HCL pH7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X‐100, 10% Glycerol, H₂O). The following reagents were used: human and mouse TNF (Enzo), zVAD‐FMK (Apex Bio), SM‐164 (gift from Shaomeng Wang), Ripk1i (GSK'963, gift from GlaxoSmithKline plc.), doxycycline (DOX) (BD Bioscience), Riboxxol (Riboxx Gmbh). The following antibodies were used: α‐RIPK1 (Cell Signaling; 3493), α‐CYLD (Cell Signaling; D1A10), α‐SHARPIN (Proteintech), α‐TRADD (BD Biosciences), α‐TNFR1 [H5] (Santa Cruz Biotechnology), α‐HSP90 (Santa Cruz Biotechnology), α‐CASP‐8—for Western blot (WB)—post‐immune‐precipitation (IP) (MBL), α‐CASP‐8—for IP [C‐20] (Santa Cruz Biotechnology), α‐Casp‐8 for rat (Cell Signalling; 9429), α‐ FADD for IP and WB [M‐19] (Santa Cruz Biotechnology), α‐Ripk3 (Pro‐science; 2283). All antibodies were used at a 1:1,000 dilution.