Immunocytochemistry
The transfected HEK-293T cells were seeded onto poly-L-lysine-treated coverslips 24 h after transfection and washed with PBS three times, then fixed with 4% paraformaldehyde at room temperature for 15 min. To reduce nonspecific binding, the cells were incubated in 4% bovine serum albumin for 1 h. The first anti-FLAG antibody (1:1000, 8146S, Cell Signaling) was added to detect the expression of the receptors, followed by incubation overnight at 4°C. The plasma membrane localization marker protein fused with GFP (mGFP) was used to label the cell membrane. After washing five times, the coverslips were incubated in a dilute solution of the fluorescent secondary antibody goat anti mouse IgG (1:500, ab150116, Abcam), usually for a few hours at room temperature (Singh et al., 2011). Photomicrographs were taken with a Leica TCS SP2 Laser Scan Inverted microscope.