For protein expression analysis, frozen tissues were homogenized in RIPA buffer containing protease inhibitors. Protein-matched samples (Bradford assay) were electrophoresed (SDS-PAGE) and then transferred to nitrocellulose (NC) membranes. The NC membranes were blocked with 5% skim milk in TBS (25 mmol/L Tris base and 150 mmol/L NaCl) for 2 h at room temperature and then incubated with the following primary antibodies (1:1,000 diluted) at 4°C overnight. SREBP1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ac-K antibody (Cell Signaling Technology, Beverly, MA, USA), ACC antibody (Thermo Fisher, Waltham, MA, USA), FAS antibody (Thermo Fisher), GAPDH antibody (Thermo Fisher), and SCD1 antibody (Abcam, Cambridge, UK). The membranes were incubated with secondary antibodies (1:5,000 diluted) at room temperature for 1 h and then washed three times for 10 min each in TBST. The target proteins were detected with ECL plus detection reagents (Amersham, Pittsburgh, PA, USA). The expression levels were quantified using optical densitometry and the ImageJ software (http://rsbweb.nih.gov).

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