Immunofluorescence Assay Protocol

Immunofluorescence assays were performed as described previously [12 (link), 47 , 48 ]. In brief, cells were plated on coverslips and fixed in 4% paraformaldehyde for 30 min, treated with 0.2% Triton X-100 in PBS for 5 min at room temperature and then blocked with 10 mM glycine. Incubation with the primary antibodies was performed for 1 h at room temperature, followed by washing in PBS and then incubation with the secondary antibodies. Photomicrographs were obtained using a confocal microscope LSM 710 (Carl Zeiss) using a 63 × 1.4/NA objective lens (Carl Zeiss). The images were acquired using the Zen 2010 software in the presence of immersion oil at room temperature.