Rabbit anti ripk3

Cells were stimulated as indicated and lysed in SDS lysis buffer with 1% β-mercaptoethanol and boiled immediately at 95 °C for 5 min. Lysates were separated by 10% or 8% SDS-PAGE and transferred to PVDF membrane. Immunoblot analysis was performed using the following antibodies: mouse anti-RipK1 (610458, BD Biosciences, Mississauga, Canada), rabbit anti-RipK3 (2283, ProSci Inc., Poway, USA), mouse anti-actin (47778, Santa Cruz Biotechnology, Dallas, USA), rabbit anti-STAT1 (9172, Cell Signaling), rat anti-caspase-8 (1G12, Enzo), rabbit anti-CIAPs (CY-P1041, Cyclex), rat anti-MLKL monoclonal (MABC604, EMD Millipore), rabbit anti-CYLD (8462, Cell Signaling), rabbit anti-A20 (5630, Cell Signaling, Danvers, USA), mouse anti-FADD (1F7, Enzo, Farmingdale, USA) and mouse anti-αTublin (5286, Santa Cruz Biotechnology, Dallas, USA). Rabbit anti-PP2A B subunit (100C1) was obtained from Cell Signaling Inc. In some cases, whole cell lysates were prepared in RIPA-EDTA free buffer supplemented with protease inhibitor and 50 Units of calf intestinal phosphatase (CIP) was used to dephosphorylate the total protein in 1× CutSmart NEB buffer for 50 min at 37 °C. SDS lysis buffer was added to the treated samples and boiled for 5 min at 95 °C followed by standard western blot protocol.