P rip1

Western blotting analysis was performed as previously described [16 (link)]. Antibodies against the following proteins were used: Cleaved-caspase-3 (1:1000, #9661), PARP (1:1000, #9542), Bcl-2 (1:1000, #15071), survivin (1:1000, #2808), p-MLKL (1:1000, #91689), MLKL (1:1000, #14993), p-RIP3 (1:1000, #93654,) RIP3 (1:1000, #13526), p-RIP1 (1:1000, #65746), RIP1 (1:1000, #3493), LC3A/B (1:1000, #12741), beclin1 (1:1000, #3495), p62 (1:1000, #5114), p-PI3K (1:1000, #4228), PI3K (1:1000, #4257), p-AKT (1:1000, #4060), AKT (1:1000, #4691), p-mTOR (1:1000, #2974), mTOR (1:1000, #2983), p-p70S6K (1:1000, #9204), and p70S6K (1:1000, #2708), all from Cell Signaling Technology (Beverly, MA, USA); Cyclin D1 (1:1000, #sc-20044), β-actin (C4, 1:1000, #sc-47778), Cdk4 (1:1000, #sc-23896), and Cdk6 (1:1000, #sc-7961), all from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and MMP13 (1:1000, NBP1-45723) from Novus Biologicals (Centennial, CO, USA). Protein bands were detected using the ProteinSimple detection system (ProteinSimple Inc., Santa Clara, CA, USA).

Tumor cells were lysed on ice with ice-cold radioimmunoprecipitation (RIPA) buffer containing a protease inhibitor cocktail (Sigma-Aldrich) for 30 min. The tumor tissues in RIPA buffer were treated by ultrasound. Then, soluble protein concentrations in lysate were determined by using a BCA protein assay kit (Thermo Scientific, Fremont, CA, USA). Western blot analysis was performed mainly as previously described [21 (link)]. The following antibodies were used: caspase-3, cleaved caspase-3, caspase-8, cleaved caspase-8 (9662S, 9661S, 4790S, 9496S respectively, Cell Signaling Technology, Danvers, MA, USA), RIP1 (610,458, BD Biosciences), pRIP1 (65746S, Cell Signaling Technology, Danvers, MA, USA), RIP3 (13,526, Cell Signaling Technology, Danvers, MA, USA), pRIP3 (ab209384, Abcam, Cambridge, UK), MLKL (ab184718, Abcam, Cambridge, UK), pMLKL (ab187091, Abcam, Cambridge, UK), JNK (9252, Cell Signaling Technology, Danvers, MA, USA), pJNK (4668, Cell Signaling Technology, Danvers, MA, USA), GAPDH (5174S, Cell Signaling Technology, Danvers, MA, USA) and corresponding secondary antibodies (Jackson ImmunoResearch, PA, USA).

Cells were lysed in buffer containing 0.5% NP-40 (Roche, 11754599001), 100 mM Tris-HCl (pH 7.4), and 300 mM NaCl and complemented with protease and phosphatase inhibitors [19 (link)]. Proteins were loaded by SDS-gel electrophoresis, and blots were probed with antibodies against glyceraldehyde 3-phosphate dehydrogenase(GAPDH) (6C5) (Millipore, NG1740950, dilution 1:5000), MLKL (D2I6N) (N° 14993, dilution 1:2500), p-MLKL (Ser358)(D6H3V) (N° 91689, dilution 1:2500), RIP1 (D94C12, dilution 1:1000) (N° 3493), prohibitin-1 (PHB1) (N° 2426S, dilution 1:1000) and p-RIP1 (Ser166)(D1L3S) (N° 65746, dilution 1:1000) from Cell Signaling, Lon peptidase 1 (LONP1) (Proteintech, 15440-1-AP, dilution 1:1000), p-RIP3 (Ser227) (EPR9627) (Abcam, ab209384, dilution 1:8000), and translocase of outer mitochondrial membrane 20 (TOMM20) (F-10) (Santa-Cruz, sc-17764, dilution 1:1000). Western blot images were analyzed by ImageJ software.