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.