Bnip3

Proteins were extracted from cells and tissues by protein extraction reagent SDS Lysis Buffer (Beyotime, Shanghai, China) and the samples were centrifuged at 10,000 g for 15 min at 4°C to collect the supernatant. With the use of the BCA protein assay kit (Beyotime, Shanghai, China), the protein concentration was determined and the protein was separated by 12.5% SDS-PAGE to transfer into PVDF membranes. The membranes were blocked for 2 h at room temperature (25°C) and incubated with different SGK1 (Abcam, Ab32374), FOXO3a (ProteinTech, 10849-1-AP), p-FOXO3a (Affinity, AF3020), BNIP3 (ProteinTech, 68091-1-Ig), Bcl-2 (ProteinTech, 26593-1-AP), Bax (ProteinTech, 50599-2-Ig), Bim (ProteinTech, 22037-1-AP), LC3 (Abcam, ab192890), Beclin-1 (Cell Signaling Technology, 3738S), p62 (ProteinTech, 18420-1-AP), and GAPDH (Santa Cruz, sc-47724) antibody overnight at 4°C. After incubation with the primary antibody, the secondary antibody was followed for 2 h at room temperature (25°C) and washed with TBST. Finally, the results were visualized by using film exposure with an ECL substrate. Relative expression was quantified with ImageJ and GAPDH was used as an internal control.

Liver tissue samples and cultured cells were lysed in RIPA buffer. The nuclear and cytoplasmic protein fractions were extracted using a nuclear and cytoplasmic protein extraction kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. Protein concentration was measured using a BCA protein assay kit (Boster, Wuhan, China). The protein samples were separated by SDS–PAGE (Bio–Rad Laboratories Inc., Berkeley, CA, USA) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corp, Billerica, MA, USA). The PVDF membranes were incubated with primary antibodies against PGC-1α, BNIP3, Beclin-1, MCAD, CPT-1α (1:500; Proteintech, Wuhan, China), PPAR-α (1:500; Cell Signaling Technology, USA), Hif-2α and LC3B (1:1000; Abcam, Cambridge, MA, USA), and Lamin A/C (1:500; Zhongshan Jinqiao, China) for 24 h, and then incubated with secondary antibody (1:500; Zhongshan Jinqiao, China) for 1 h at room temperature. The protein bands were visualized by enhanced chemiluminescence (ECL) assay (Thermo Scientific, USA), and the blots were analyzed using Image Lab 3.0 (Bio–Rad, Hercules, CA, USA). The gray value of protein bands was calculated using Image J 1.50i (National Institutes of Health, USA) and normalized to that of the internal control, β-actin.