Western blot analysis of autophagy- and apoptosis-related protein expression in PC cells

Following treatment, the cells exposed to phosphatase inhibitors were collected, total protein was extracted, the concentration was measured in all samples using the Bradford Protein Assay kit, and the protein samples (30-100 µg) were separated using 10-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were then transferred onto a polyvinylidene fluoride membrane, which was blocked for 1.5 h in blocking buffer consisting of 5% milk in Tris-buffered saline (TBS) containing 0.05% Tween-20, and then incubated with the different primary antibodies (1:400) overnight at 4°C. The membrane was washed with TBS containing 0.1% Tween-20, incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:1,000) for 1 h at 25°C. Primary and secondary antibodies were diluted in Tris-buffered saline (TBS) containing 0.05% Tween-20. The immunoreactive bands were then visualized using enhanced chemiluminescence kits. The density of the immunoreactive bands was analyzed using the Image J software (v1.8.0; National Institute of Health).