Fibronectin coated 8 chamber glass slides

6 × 10⁴ HEK293 cells per well were seeded into fibronectin-coated 8-chamber glass slides (ThermoFisher Scientific) and incubated for 24h. Cells were transfected with 4 ng indicated expression construct for 48h and fixed with 4% formaldehyde in PBS for 15 min. Fixed cells were washed three times with PBS (5 min each), blocked with 5% normal goat serum in PBS for 1h, and incubated with rabbit αFLAG antibody (Sigma-Aldrich, 10 μg/ml) in PBS/1% BSA for 1h. Cells were washed as before and incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch, 1:1000) in PBS/1% BSA for 1h. Cells were washed as before and coverslips mounted using VECTASHIELD HardSet Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). Stained cells were imaged using a fluorescence microscope with Apotome (Zeiss, Oberkochen, Germany).