Flip l

Western blot analysis was performed as previously described42 . DR4 antibody was from Novus (Novus Biologicals, Littleton, CO, USA); DR5 and P62 antibodies were from Abcam (Cambridge, UK); FLIP-L and FLIP-S antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Caspase 3, Caspase 8, Caspase 9, PARP, CHOP, PERK, p-GCN2, GCN2, p-PKR, PKR, HRI, p-eIF2α, eIF2α, ATF4, MAP1LC3B, tubulin, GAPDH and β-actin antibodies were from Cell Signaling Technology (Beverly, MA, USA); The secondary antibodies used were HRP-conjugated anti-mouse IgG and anti-rabbit IgG from sigma (USA).

The MKN28 human gastric carcinoma cells obtained from the American type culture collection (Manassas, VA, USA) were cultured in RPMI medium (Invitrogen Corp, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (GIBCO BRL, Grand Island, NY, USA), 1 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. Antibodies against TRAIL, DR4, DR5, Fas, FasL, XIAP, a cellular inhibitor of apoptosis protein-1 (cIAP-1), cIAP-2, caspase-3, -8, -9, Bcl-2, Bax, Flip_L, Flip_S, PARP, β-catenin, and PLCγ1 were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). An antibody against β-actin was from Sigma (Beverly, MA, USA). Caspase activity assay kits were obtained from R&D Systems (Minneapolis, MN, USA). An enhanced chemiluminescence (ECL) kit was purchased from Amersham (Arlington Heights, IL, USA). 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) were obtained from Calbiochem (San Diego, CA, USA). All other chemicals not specifically cited here were purchased from Sigma–Aldrich (St. Louis, MO, USA). All these solutions were stored at −20 °C. Stock solutions of 4,6-diamidino-2-phenylindole (DAPI, 100 μg/mL) and propidium iodide (PI, 1 mg/mL) were prepared in phosphate-buffered saline (PBS).