Phos s6

The ECC-1 and KLE cells treated with ONC201 for 24–36 h. Cell lysates were prepared in RIPA buffer. Protein concentration was measured by BCA assay. Equal amounts of protein were separated by 10–12% gel electrophoresis and transferred onto a PVDF membrane. The membrane was blocked with 5% nonfat dry milk and then incubated with different primary antibodies overnight at 4 °C. The primary antibodies from Cell Signaling (Beverly, MA) used in this study included as follows: MCL-1, BCL-2, Phos-S6, Pan-S6, Phos-AKT, Pan-AKT, PERK, Bip, Erol-1, IRE1-a, PD-I, CDK4, CyclinD1, E-cadherin, VEGF and Slug. DRD2 and DRD5 antibodies were from Santa Cruze Biotechnology Inc. (Dallas, TX).). β-Actin and α-Tubulin were from Sigma (St. Louis, MO). β-Actin and α-Tubulin were used at 1:5000 dilution, and the rest antibodies were diluted: 1:1500. The membrane was then washed and incubated with a secondary peroxidase-conjugated antibody for 1 h after washing. Antibody binding was detected using SuperSignal™ West Pico on the ChemiDoc™ Image System (Bio-Rad). After developing, the membrane was stripped and re-probed using antibodies against β-actin or α-tubulin to confirm equal loading. Intensity for each band was measured and normalized to β-actin or α-tubulin as an internal control.