Anti rip2

Cells were lysed with RIPA lysate to obtain total protein. Total proteins were separated by electrophoresis on 15% SDS‐PAGE and then transferred onto a PVDF membrane of 0.2 µm pore size (Millipore) for Western blot analysis. The PVDF membrane was blocked with 5% skim milk powder in Tris‐buffered saline/1% Tween‐20 (TBS‐T) for 2 hours at 4°C and then incubated with the primary antibodies anti‐RIP2 (monoclonal, rabbit anti‐human, 1:1000), anti‐NF‐κB p65 (monoclonal, rabbit anti‐human, 1:2000), anti‐p‐NF‐κB p65 (monoclonal, rabbit anti‐human, 1:8000), anti‐IκBα (monoclonal, mouse anti‐human, 1:1000), anti‐MGMT (monoclonal, rabbit anti‐human, 1:1500), anti‐LaminB (monoclonal, rabbit anti‐human, 1:1000), and anti‐GAPDH (monoclonal, rabbit anti‐human, 1:1000) (Cell Signaling Technology) overnight at 4°C. Afterward, the membranes were washed thrice with TBS‐T for 10 minutes and were incubated with horseradish peroxidase (HRP)‐labeled secondary antibodies of sheep to mouse and sheep to rabbit (ZSGB‐BIO Co., Ltd.) at 4°C for 1 hour. The non‐binding secondary antibodies were washed out thrice by TBS‐T for 10 minutes. Protein bands were detected using a SuperSignal West Femto Substrate (Thermo Fisher) and detected using a GE AI600 system (GE). Densitometric analysis of protein bands was performed using ImageQuant TL7.0 (GE).

Cells were collected in centrifuge tubes and lysed with RIPA lysis buffer (Beyotime) to obtain total protein. The total protein concentration was measured in a BCA protein quantification kit (Beyotime), and a sample containing 3 μg/μl total protein was prepared. Proteins were separated by 10% SDS‐PAGE electrophoresis and transferred to 0.22 μm pore size PVDF membranes (Millipore, Billerica, MA, USA). PVDF membranes were blocked with 5% BSA (Beyotime) for 4 h and incubated with primary antibodies anti‐RIP2 (4142S, 1:1000), anti‐NF‐κB p65 (8242S, 1:2000), anti‐p‐NF‐κB p65 (3033S, 1:8000), anti‐IκBα (4812S, 1:1000), anti‐SOX‐2 (4900S, 1:1000), anti‐CD133 (64326S, 1:1000), and anti‐GAPDH (5174S, 1:1000) (Cell Signaling Technology, Danvers, MA, USA) overnight. Unbound primary antibody was washed away, and the PVDF membrane was labeled with horseradish peroxidase (HRP) goat anti‐mouse antibody (7076S, 1:3000) or goat anti‐rabbit antibody (7074S, 1:3000) (Cell Signaling Technology) and incubated for 1 h. Unbound secondary antibodies were washed away, and PVDF membranes were developed with SuperSignal West Femto Substrate (ThermoFisher, Waltham, MA, USA) and imaged using a GE AI600 system (GE, Chicago, IL, USA). Densitometric analysis of protein bands was performed using ImageQuant TL7.0 (GE).

Anti‐RIP1 (#3493), anti‐RIP2 (#4142), anti‐RIP3 (#13526), anti‐p‐RIP1 (#44590), anti‐MLKL (#14993), anti‐p‐MLKL (#91689), anti‐caspase‐8 (#4790), anti‐cleaved caspase‐8 (#9496), anti‐PARP (#9532), anti‐GSDMD (#96458), anti‐caspase‐8 (#9746), anti‐Myc 9B11 (#2276), anti‐GFP (#2956) (Cell Signaling Technology), anti‐actin (#MAB1501, MILLIPORE), anti‐IL18 (PM014, MBL), and anti‐FLAG antibodies (F7425, Sigma‐Aldrich) were obtained from the indicated suppliers. The following inhibitors, all obtained from Calbiochem, were used at the indicated final concentrations: z‐VAD‐FMK, 10 μM; RIP1 kinase inhibitor III, 5 μM; GSK872 (RIPK3 inhibitor), 5 μM; and caspase‐8 inhibitor II, 10 μM. Staurosporine was purchased from Sigma.