Cells were harvested 48 h after transfection and homogenised using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific, United States). Proteins extracted from the transfected cells were blotted onto a polyvinylidene difluoride membrane and incubated overnight at 4°C with an anti-FLAG antibody (1:5,000 dilution, Abways, Shanghai, China). The membrane was incubated with secondary antibodies (goat anti-mouse IgG; 1:5,000 dilution; Abways, Shanghai, China) on the following day. Finally, blots were developed using an ECL western blotting kit (Pierce Biotechnology, Rockford, IL, United States).
Immunofluorescence analysis was performed on transfected Chinese hamster ovary (CHO) cells grown on coverslips using anti-FLAG (1:500 dilution) primary antibodies. Fluorescent images were captured using a confocal microscope (Olympus FV1000, Tokyo, Japan).
Immunofluorescence analysis was performed on transfected Chinese hamster ovary (CHO) cells grown on coverslips using anti-FLAG (1:500 dilution) primary antibodies. Fluorescent images were captured using a confocal microscope (Olympus FV1000, Tokyo, Japan).