Protein Extraction and Western Blot Analysis

Total tissue protein was extracted using RIPA lysis buffer and protease inhibitor phenylmethane sulfonyl fluoride (Merck Millipore, Burlington, MA, USA). A BCA protein assay kit (Beyotime Biotechnology, Shanghai, China) was used to measure the amount of protein in the sample, in accordance with the manufacturer’s recommendations. Equivalent amounts of protein (20 μg) were separated on an SDS PAGE gel and then transferred onto 0.45 μm PVDF membranes (Millipore, Billerica, MA, USA) according to standard protocols. Following established procedures, equivalent protein concentrations (20 μg) were separated on an SDS PAGE gel and then deposited onto 0.45 μm PVDF membranes from Millipore (Billerica, MA, USA). After being blocked with 5% milk in TBST buffer for 1 h at room temperature, the membranes were incubated with primary antibodies for an overnight period at 4 °C. Proteins were identified using ECL reagent following secondary antibody incubation for 1 h at room temperature (Millipore Billerica, MA, USA). Three independent replicates were performed for each [21 (link)].