Western blotting

To prepare cell lysates, the cells were washed with ice-cold PBS and homogenized using a Bullet Blender (Next Advance, Averill Park, NY, USA) in PLC lysis buffer (30 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 1 mM EGTA, 10 mM NaPPi, 100 mM NaF, 1 mM Na₃VO₄) supplemented with protease inhibitor cocktail (Roche), 1 mM PMSF.^2,31 Total protein concentrations of the lysates were determined using the DC protein assay (Bio-Rad). Western blotting and image analysis were conducted as described previously.^2,31 Antibody information: GAPDH (MA5-15738) and β-actin (MA5-15739) antibodies from Pierce (Rockford, IL, USA); antibodies against FoxO1 (9454 s), phospho-FoxO1 (Thr24) antibody (9464 s), LC3 (2775 s) and p62 (SQSTM1, 5114 S) from Cell Signaling Technology (Beverly, MA, USA); Tfeb (A303-673 A) antibody from Bethyl Laboratories, Inc. (Montgomery, TX, USA).