Ab280376

Primary antibodies targeting LC3B (1:1000; Abcam, ab221794, 14, 16 kDa), Atg5 (1:1000; Abcam, ab108327, 55 kDa), Atg7 (1:1000; Abcam, ab133528, 77 kDa), p62 (1:1000; Abcam, ab109012, 62 kDa), ALKBH5 (1:1000; Abcam, ab195377, 44 kDa), p-GSK3β (Ser9, 1:1000; Abcam, ab107166, 47 kDa), GSK3β (1:1000; Abcam, ab280376, 46 kDa), p-mTOR (S2448, 1:1000; Abcam, ab109268, 289 kDa), mTOR (1:1000; Abcam, ab134903, 289 kDa), and Beclin-1 (1:1000; Abcam, ab207612, 52 kDa) were used. Horseradish peroxidase (HRP)-linked anti-mouse IgG and anti-rabbit IgG (1:5000; Protein Tech Group, SA00001-2) were used, respectively. Data were normalized to β-actin expression. Immunoreactive bands were quantified by densitometry and computer-assisted image analysis.

Pierce™ Classic Bead Co-IP Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for Co-IP assay according to the manufacturer's instructions. After transfection, the 293T cells were washed with cold PBS, added 1 mL of PBS, and scraped the cells to a 1.5 mL EP tube, and draw 100 μL from it as input. Then, the lysis buffer containing complete protease inhibitor was added to the remaining cell suspension, centrifuged at 14,000 × g at 4°C for 20 min. The supernatant was incubated with normal IgG (1 : 50, ab172730, Abcam) or anti-GSK3B (1 : 30, ab280376, Abcam) antibody. Then, the immune complex was combined with protein A-sepharose and analyzed by western blot.