Western Blot Analysis of Proteins

Cells were lysed in IP buffer (50 mM Tris HCl (pH 7.5), 150 mM NaCl, 1% NP40, 2 mM Sodium orthovanadate, 10 mM NaF, 10 mM β-glycerophosphate, 1 mM PMSF) [15 (link)]. Proteins in lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to PVDF membrane (Millipore). Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG, sheep anti-mouse IgG, and donkey anti-goat IgG were used as secondary antibodies. Chemiluminescence detection was performed using WEST-ZOL plus (iNtRON) and West Femto Substrate (Thermofisher). X-ray films (Agfa) and LAS (Image Quant; LAS4000) were used to visualize the chemiluminescence signal. 0.1% TBS-T was used for washing and blocking solution preparation, and 0.1% PBS-T was used for LC3B blotting [51 (link), 52 (link)].