Lds sample buffer

Protein samples were extracted by lysing glioma cells in RIPA buffer containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MI, USA; Cat. # P8340) and 1 mM PMSF. Subsequently, 30 µg of proteins were denatured using 100 mM dithiothreitol and LDS Sample Buffer (Abcam, Cambridge, UK; Cat. # ab119196) and heating. The proteins were separated electrophoretically and transferred onto a PVDF membrane (Merck Millipore, St. Louis, MI, USA; Cat. # IPVH00010). The membranes were blocked with 5% non-fat milk in TBST for 1 h and incubated overnight at 4 • C with primary antibodies, including the Total OXPHOS Human WB Antibody Cocktail (Abcam, Cat. # ab110411), TOMM20 (Abcam, Cat. # ab186735), beta-actin (GenScript, Piscataway, NJ, USA; Cat. # A00730), Ser473-phospho-Akt (Cell Signaling Technology, Danvers, MA, USA; Cat. # 9271), Thr308phospho-Akt (Cell Signaling Technology Cat. # 9275), Akt (Cell Signaling Technology Cat. # 9272), Thr172-phospho-AMPKα (Cell Signaling Technology Cat. # 2535), and AMPKα (Cell signaling technology Cat. # 2532). Following this, membranes were washed in TBST and treated with HRP-conjugated secondary antibodies (GE Healthcare, Chicago, IL, USA). Protein detection was achieved by inducing chemiluminescence with Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA; Cat. # 1705061) and visualized using the ChemiDoc XRS+ system (Bio-Rad)