4.4. Western Blot Analysis

INS-1 cells and C2C12 cells were plated in 6-well plates and cultured overnight, then treated with schisandrin C for 24 h. Subsequently, the cells were lysed with RIPA buffer (Cell Signaling, Danvers, MA, USA) for 20 min. Equal amounts of protein were separated by size using 10% sodium dodecyl sulfate-polyacrylamide gel [38]. Separated proteins were transferred by electroblotting to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were probed with the primary antibodies (Cell Signaling) overnight at 4 °C, then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibodies (Cell Signaling) for 1 h at 4 °C, and with enhanced chemiluminescence reagent (GE Healthcare UK Limited, Buckinghamshire, UK) for 5 min at room temperature. Proteins were detected using a chemiluminescence system (FUSION Solo, PEQLAB Biotechnologie GmbH, Erlangen, Germany).