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.