Confocal Microscopy Analysis of Transfected Cells

Confocal laser scanning microscopic observation was conducted as previously described [23 (link)]. In brief, 48 h after transfection, 293A cells were fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were then treated with a fluorescent wheat germ agglutinin conjugate (WGA, Alexa Fluor^® 594 conjugate; Thermo Fisher Scientific). After 0.02% (w/v) Triton-X100 treatment, the nuclei of the cells were visualized with TO-PRO-3 Iodide (Molecular Probes, Eugene, OR, USA). Stained cells were mounted with VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, USA), after which fluorescence was detected using an FV10i Confocal Laser Scanning Microscope (Olympus, Tokyo, Japan).