Western Blot Analysis of Cell Signaling

Cells were appropriately treated, and total cell lysates and nuclear extracts were prepared as previously described [35 (link)]. Total protein lysates from cells were prepared by sonicating cells in lysis buffer (iNtRON, Korea). Equal amounts of lysates were boiled at 100°C and resolved by 10-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA) and then blocked with 5% milk in Tris-buffered saline and 0.1% Tween 20. The membranes were incubated overnight with primary Abs against LC3, AMPKα, phosphorylated AMPKα, p70S6K, phosphorylated p70S6K, and β-actin (all from Cell Signaling Technology, Danvers, MA), and proteins were detected with goat-anti-rabbit Abs conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology, Danvers, MA). Proteins were detected using the enhanced chemiluminescence (ECL) method with femtoLUCENT ™ PLUS-HRP (G-biosciences, St. Louis, MO).