Anti p rip1

Samples from tissues or cells were lysed in RIPA buffer supplemented with 1x protease and phosphatase inhibitor cocktail (Thermo Fisher, America) and incubated on ice for 30 min. After centrifugation at 14000 × g for 10 min at 4 °C, the supernatant was collected to determine the protein concentration by BCA assay. The samples were prepared for electrophoresis, followed by transfer onto PVDF membranes. The primary antibodies used in this work were as follows: anti-ZO-1 (Santa Cruz Biotechnology), anti-p-MLKL (Cell Signaling Technology), anti-p-RIP1 (Cell Signaling Technology), anti-cleaved/full-length caspase 9 (Cell Signaling Technology), anti-cleaved/full-length caspase 3 (Cell Signaling Technology), anti-GPX4 (Santa Cruz Biotechnology), anti-SLC3A2 (Santa Cruz Biotechnology), anti-SLC7A11 (Santa Cruz Biotechnology), anti-ANXA2 (ABclonal), anti-His-tag (ABclonal), anti-HA-tag (ABclonal), anti-HSPB1 (Cell Signaling Technology), and anti-PRDX1 (Cell Signaling Technology). Secondary antibodies were purchased from ABclonal. The membranes were visualized via the chemiluminescence method.

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.

Antibodies used in immunoblotting and immunofluorescence: anti-RIP3 (Cell Signaling Technology, 13526s, 1:1000), anti-p-RIP3 (Abcam, ab209384, 1:1000), anti-MLKL (Abcam, ab184718, 1:2000), anti-p-MLKL (Abcam, ab187091, 1:1000), anti-mouse p-MLKL (Abcam, ab196436, 1:5000), anti-RIP1 (BD Biosciences, 610458, 1:1000), anti-p-RIP1 (Cell Signaling Technology, 65746, 1:1000), anti-mouse p-RIP1 (Cell Signaling Technology, 31122, 1:1000), anti-ACTIN (Sigma-Aldrich, A3853, 1:5000), anti-p-ERK (Cell Signaling Technology, 9101s, 1:1000) and anti- IκB-α (Santa Cruz Biotechnology, sc-371, 1:5000). TNF-α and zVAD were purchased from R&D Systems. SMAC mimetic (LCL-161) was purchased from Adooq Bioscience. Dabrafenib and GSK’872 were purchased from Selleckchem. Necrostatin-1, lipopolysaccharide (LPS), and propidium iodide (PI) were purchased from Sigma-Aldrich. Cycloheximide was purchased from Calbiochem. Polyehylenmine was purchased from Polysciences. We purified GST-TRAIL.