Western Blot Analysis of Protein Samples

The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA) using a Bio-Rad semidry blotter (Bio-Rad, USA). The membranes were probed using the method previously described (55 (link)). In short, the membrane was blocked in 20 mM Tris-buffered saline with 0.05% Tween 20 (TBST) (pH 8.0) in 5% skim milk for 1 h at room temperature and then incubated with the primary antibody for 1 h or for 12 h at 4°C. Subsequently, the treated membrane was washed three times with TBST for 15 min each before incubating with a secondary antibody for 1 h at room temperature. After three more washes in TBST for 15 min each, the immune-reactive proteins could be localized. The membranes were treated with Clarity Western ECL substrate (number 170-5061; Bio-Rad, USA) for 5 min under dark conditions. The primary antibody used is monoclonal mouse anti-FLAG (number M20008S; Abmart, Shanghai, China) at a dilution of 1:5,000. The secondary antibody used is goat anti-mouse secondary antibody (number M21001L; Abmart, Shanghai, China) at a dilution of 1:5,000. Immune-reactive bands were visualized by the VIBER molecular imaging system (Fusion FX7, France).