Alexa fluor 488 or 594 conjugated anti rabbit or anti mouse secondary antibodies

For immunofluorescence assay, the transfected cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton x-100 in phosphate-buffered saline (PBS). They were then blocked with the blocking buffer (5% goat serum and 2% BSA in 1× PBS) for at least 30 min at RT. Subsequently, the cells were incubated with appropriate primary antibody at 4 °C overnight. The next day, after a total of three times washing in phosphate-buffered saline with 0.05% Tween 20 (PBST), the stained proteins were set with Alexa Fluor 488- or 594-conjugated anti-rabbit or anti-mouse secondary antibodies (Molecular Probes, Seoul, Korea). Nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, MO, USA). They were then pictured using an Olympus Ix71 fluorescence microscope (Olympus, Seoul, Korea).

Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X‐100 in phosphate buffered saline (PBS). Following fixation, cells were incubated at 4 °C overnight with a primary antibody in PBS with 1% bovine serum albumin and 0.1% Triton X‐100. Stained proteins were visualized using Alexa Fluor 488‐ or 594‐conjugated anti‐rabbit or anti‐mouse secondary antibodies (Molecular Probes, Seoul, Korea). Nuclei were counterstained with DAPI (Sigma‐Aldrich). Stained cells were observed by using an Olympus IX71 fluorescence microscope (Olympus, Seoul, Korea).