Immunofluorescence
Fibroblast and macrophage cells were transfected with pFLAG-SHARPIN. 48 hours later, cells were washed briefly with PBS, then fixed in cold methanol at −20°C for 10 minutes and cold acetone at -20°C for 1 minute. Incubate cells with PBS+1% BSA for 15 minutes to block non-specific binding. Fixed cells were then incubated with anti-FLAG (1∶100) as the primary antibody at room temperature for 1 hour, followed by three 5-minute washes. Cells were further incubated with goat anti-rabbit IgG-FITC (1∶100) as the secondary antibody at room temperature for 30 minutes, followed by three 5-minute washes. Cells were then examined using inverse fluorescence microscope.