Immunofluorescent Staining of Cultured Cells

Immunofluorescent staining was performed as previously described32 (link). In brief, the cells grown on CultureSlides (BD Biosciences) were exposed to the indicated reagents, fixed with 4% paraformaldehyde, washed twice with ice-cold PBS, permeabilised with 0.1% Triton X-100, and then blocked with 1% BSA for 1 h at room temperature. Subsequently, the cells were incubated with the indicated primary antibody for 16 h at 4 °C. After washing with PBS to remove unbound primary antibodies, bound antibodies were visualised by incubating the cells with secondary antibodies (Molecular probes). 4′,6-Diamidino-2-phenylindole (DAPI) was used as a counterstain to visualise the nuclei. The fluorescent images were obtained using a Leica DMI 4000B microscope with appropriate filters and lasers. For confocal analysis, the images were obtained using a FV1000 confocal microscope (Olympus) with a 60 × oil immersion lens, NA 1.35 (Uplsapo).