Anti cdk2

Cells were lysed with radio immunoprecipitation assay buffer (RIPA) containing 1 mmol/L PMSF (Solarbio, China). Protein extractions were separated by 12% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Germany). The membranes were incubated overnight at 4 °C with primary antibodies specific for anti-β-actin, anti-CDK2, anti-CyclinD1, anti-CyclinE, anti-PCNA, and anti-P53 (1:1,000; Wanleibio), anti-Bcl-2, anti-Bax, anti-Caspase9, anti-FOXC1, and anti-MyoD1 (1:1,000; Abways Technology), anti-MyoG, (1:1,000; Abcam, Cambridge, England). After incubation with secondary antibodies, signals were detected using a chemiluminescence system (Bio-Rad, USA).

Total proteins of bovine fat cells were extracted using radioimmunoprecipitation assay buffer (RIPA) buffer with 1% PMSF (Solarbio, Beijing, China) after transfection for 24 h or induction for 6 days after transfection. The protein concentration was determined by a bicinchoninic acid (BCA) kit (Beyotime, Shanghai, China), a 5× protein loading buffer (containing mercaptoethanol) was added to proteins at a ratio of 1:4, and then the sample was heated in boiling water for 3–5 min. Proteins were then separated by SDS-polyacrylamide gel electrophoresis and subsequently transferred to nitrocellulose membranes and blocked with milk powder solution for 2 h at room temperature. The membranes were then incubated overnight with the primary antibody of anti-CDK2, anti-PCNA, anti-PPARγ, anti-C/EBPβ, and anti-β-actin (Wanlei Bio, Shenyang, China). The membranes were then washed with PBS-Tween 20 and incubated for 1.5 h with horseradish peroxidase-conjugated secondary antibodies (Boster, Pleasanton, CA, USA). Finally, the membranes were imaged with an enhanced chemiluminescence (ECL) kit (Solarbio, Beijing, China) and quantified with the ImageJ program (Bio-Rad, USA).