Protein Extraction and Western Blot Analysis

Vero cells were lysed in lysis buffer containing 50 mM Tris-HCl (pH 6.8), 10% glycerol, and 2% SDS [20 (link)]. The protein concentration was quantified by the BCA protein assay kit and equal amounts of protein samples were mixed with 5× sample loading buffer and boiled for 10 min, and then separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were electro-transferred to 0.45 μm PVDF membranes (Millipore, Mississauga, ON, Canada). Membranes were blocked with 5% (w/v) skim milk-TBST at room temperature for 2 h and then incubated overnight at 4 °C with primary antibodies. The blots were then incubated with corresponding horseradish peroxidase (HRP) conjugated secondary antibodies (ABclonal, Wuhan, China). The protein bands were visualized using the Clarity™ Western ECL Blotting Substrate (Bio-Rad, Hercules, CA, USA). The protein blots were quantified by Image J software (National Institutes of Health, Bethesda, MD, USA).