Xiap 3b6

ATG2A (#15011), ATG5 (#2630), ATG7 (#2631), BECN1 (#3738), BIRC2 (D5G9), CASP8 (1C12), FADD (#2782), FAS (C18C12), FASLG (#4273), HSP90 (C45G5); LAMP1 (C54H11), MLKL (D2I6N), PARP1 (46D11), RIPK1 (D94C12), RIPK3 (D4G2A), ULK1 (D9D7), and XIAP (3B6) antibodies were obtained from Cell Signaling. SQSTM1 (#ab56416) and STX17 (#ab116113) antibodies were purchased from Abcam. β-actin (ACTB; AC-74) and ATG2B (#HPA019665) were purchased from Sigma. MAP1LC3B (#NB100-2220) was purchased from Novus Biologicals. Cell lysis, co-immunoprecipitation, subcellular fractionation, and western blotting were performed as previously described^{19 (link),41 (link)}. Relative densities of the target bands to the reference bands was calculated using ImageJ (NIH).

ACH-2 cells (1 × 10⁶ cells/ml) were treated with different concentrations of the drug for 24 h, and the cells were washed twice with PBS. Cells were homogenized by CelLytic M Cell Lysis Reagent (Sigma-Aldrich, St. Louis, MO, United States) containing a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and were incubated at room temperature (RT) for 15 min. The cells were then centrifuged at 13,000 rpm for 15 min, and the supernatant of the whole cell lysate was collected. The bicinchoninic acid (BCA) protein assay (Thermo Scientific, Rockford, IL, United States) was used to measure the actual protein amount in the treated and untreated cells. The protein extracts were separated through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then were blotted onto a Trans Blot Turbo polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, United States). Protein bands were visualized using 3,3′,5,5′-tetramethylbenzidine (TMB) solution (Wako Pure Chemical). The sources of the antibodies were as follows: c-IAP1 (D5G9), c-IAP2 (58C7), and XIAP (3B6) were purchased from Cell Signaling Technology (Danvers, MA, United States); β-actin (C4) was purchased from Santa Cruz Biotechnology.