α ripk3

The following reagents were used: zVAD-FMK (10 μM, Apex Bio), QVD (10 μM, Apex Bio), SM-164 (100 nM, gift from Shaomeng Wang), GSK’963 (referred as RIPK1i, 100 nM, GlaxoSmithKline), Nec-1 (10 μM Merck), Nec-1s (10 μM Merck), RO-3306 (9 μM, Merck), Thymidine (Sigma), BIM peptide (Kind gift of Tony Letai), FLAG-hTNF (10 ng/ml, Enzo), MG132 (1-20 μM, as indicated per cell line, see below, SIGMA), Recombinant Casp8 (1U Enzo). The following antibodies were used for western blotting: α-RIPK1 (1:1000, BD Biosciences), α-HA (1:1000, Roche), α-PLK1 (1:1000, Bethyl Laboratories), α-PLK1-pT210 (1:2000, AbCam), α-Cyclin B (1:1000, Cell Signaling), α-BUBR1 (1:1000, BD Bioscience), α-BUBR1-pT680 (1:1000, Kind gift of Geert J.P.L Kops), α-BUBR1-pT676 (1:1000, Kind gift of Erich A. Nigg), α-pH-H3 (1:2000, Millipore), α-Casp8 (for WB - post IP, 1:5000, MBL), α-Casp8 - for IP (7.5 μg/ml, C-20, Santa Cruz Biotechnology, α-FADD – for IP (7.5 μg/ml, Santa Cruz), α-FADD (1:1000 BD Biosciences), α-RIPK3 (1:1000 Proscience), α-RIPK3 (1:1000 Novus Biological) α-cFLIP (1:1000, Enzo), α-Myc (1:1000, clone 9E10,SIGMA), α-HSP90 (1:1000 Santa Cruz Biotechnology).

Protein lysates were isolated from liver samples as described previously [9 (link)]. Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membrane and analyzed by immunoblot with the following antibodies: α-NEMO, α-β-actin (Sigma Aldrich, St. Louis, MO, USA), α-cleaved Caspase-3, α-cleaved Caspase-8, α-IKKβ, p-JNK, JNK1 (Cell Signaling Technology, Danvers, MA, USA), α-IKKα, α-RIPK3 (Novus Biologicals, Centennials, CO, USA), α-PCNA (Thermo Fisher Scientific, Waltham, MA, USA), and α-GAPDH (Bio-Rad Laborories, Hercules, CA, USA). As secondary antibodies, α-rabbit-HRP, and α-mouse-HRP (GE Healthcare, Little Chalfont, UK) were used. Densitometry was performed by using ImageJ software (ImageJ, NIH, Bethesda, MD, USA).