Ep1793y

Cells cultured on glass coverslips were rinsed several times in Ca²⁺- and Mg²⁺-containing PBS, fixed in 4% paraformaldehyde (PFA) for 10 min at 37°C, and permeabilized in 4% PFA containing 0.1% Triton X-100 for 1 min at room temperature. After blocking in 4% Block Ace solution (Yukijirushi Inc., Sapporo, Japan), the cells were incubated for 1 hr separately with primary antibodies: OB-cadherin (#4442; Cell Signaling Technology, Beverly, MA, USA) at 1:100 dilution; and EPLIN (16639-1-AP; Proteintech, Rosemont, IL, USA), β-catenin (H102 sc-7199; Santa Cruz, Dallas, TX, USA) and α-catenin (anti-α1 catenin antibody EP1793Y; Abcam, Cambridge, UK) all at a dilution of 1:50. After washing, bound antibodies were detected using Alexa Fluor 488-labeled anti-rabbit secondary antibody (Thermo Fisher Scientific) for 30 min. Then, F-actin (Phalloidin, Thermo Scientific) was stained in all slides. Cell nuclei were stained with DAPI (SlowFade Gold Antifade Mountant with DAPI; Thermo Fisher Scientific). As a negative control, normal rabbit IgG was used at the same concentration instead of the primary antibody in each experiment. Microphotos were taken using a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan).