Immunofluorescence analysis
Cells (2×105) were fixed with 4% paraformaldehyde for 30 minutes at room temperature, and immunolabeled as previously described (Briguglio et al., 2013, Bowman and Turkewitz, 2001). Briefly, cells were first incubated for blocking with 5% BSA (bovine serum albumin, BP1600-1 Fisher Scientific) in PBT (0.3% Triton X-100 (ACROS) in PBS) for one hour at room temperature and then incubated with the primary antibodies in the same blocking buffer. Anti-FLAG M2 antibody staining was at 1:1000 (Monoclonal ANTI-FLAG M2 antibody, Sigma F1804) for overnight at 4°C, followed by three washes in 0.3% Triton X-100 in PBS. The secondary antibodies were Alexa Fluor 488-conjugated anti-mouse IgG, incubated for one hour at room temperature (1:1000, Alexa Fluor 488 Goat anti-Mouse IgG, Invitrogen A-11001), and similarly washed. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (50 ng ml-1). Sample was mounted on microscopic slides for imaging.