Quantitative Protein Expression Analysis

As previously described with modification [1 (link)], the cells and tissues were collected and lysed in Radio-Immunoprecipitation Assay (RIPA) buffer (Thermo Fisher, Rockford, IL, USA), containing 1% phenylmethylsulfonyl fluoride (PMSF). Protein concentration was quantified with the BCA Protein Kit (Beyotime, China). Lysates (10 μg proteins) were separated by 10% SDS-PAGE gels and transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% Bovine Serum Albumin in TBS/Tween20 (TBST) for 2 hours at room temperature, and then probed with primary antibodies against proteins of interest overnight at 4°C. After incubated with secondary antibodies for 1 hour at room temperature, protein signal was detected with the ECL chemiluminescent detection system (Bio-Rad), and protein levels were normalized to β-actin.