Las 4000 film

Whole-cell lysates were prepared by incubating cells in RIPA buffer (Beverly, MA, USA) supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, cells were harvested and collected by centrifugation at 13,200 rpm for 5 min and washed in Dulbecco’s Phosphate-Buffered Saline (DPBS) (pH 7.2). The pellets were solubilized in the same volume of mitochondrial lysis buffer for 5 min and then centrifuged at 13,200 rpm for 20 min at 4°C. Equal amounts of lysate protein were loaded and separated on a 15% SDS-PAGE gel. Proteins were electrophoretically transferred to a PVDF membrane, and the membrane was blocked in 5% skim milk in tris-buffered saline containing 0.1% Tween-20 for 1 h. Then, the membranes were incubated in the presence of following primary antibodies (LC3, beclin-1, mTOR, AMPK, GAPDH from cell signaling (Beverly, MA, USA); GATE16, TP53INP2/DOR from Epitomics (Burlingame, CA, USA)) at 4°C overnight. The membranes were then washed three times with TBS Tween 20 and probed with the corresponding secondary antibodies conjugated with HRP at room temperature for 1 h. Detection was carried out using an enhanced ECL Advance Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, UK) followed by LAS-4000 film (Fujifilm, Tokyo, Japan).