Western Blot Analysis of Protein Expression

BPC lysed by RIPA Lysis and Extraction Buffer (ThermoFisher Technology, Beijing, China). Protein content was determined by the BCA protein assay reagent (Betotime Biotechnology, China), and 20 μg of each sample was subjected to polyacrylamide gel electrophoresis. The separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes under 200 mA for 90 min. PVDF membranes were incubated with primary antibody (1:1000 dilution; PAB12542, Abnova Diagnostics, Dongguan, China), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution; Zymed, San Diego, CA, USA). The membrane was re-probed with a primary antibody against GAPDH (1:3000 dilutions; Santa Cruz Biotech., Santa Cruz, CA, USA) as a control^{56 (link)}. The assay was repeated to confirm the result.