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The membrane was washed three times with Tris-buffered saline containing Tween 20 (TBST), soaked in blocking buffer for 1 h at RT, and incubated overnight with primary antibodies for AMPK (4150), p62 (39,786), LC3 (2775), and -actin (4970) (Cell signaling technology, Beverly, MA, USA) diluted with TBST and 5% bovine serum albumin (BSA)