Western Blot Analysis of Apoptosis and Signaling

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).