Anti nectin 1

HCECs were cultured on glass coverslips until 70% to 90% confluence and then fixed in 4% paraformaldehyde for 20 minutes. Next, cells were permeabilized with 0.03% Triton X-100, blocked with 3% bovine serum albumin, and stained with primary antibodies (anti–NECTIN-1, 1:100; Abcam, Cambridge, UK). Then, the samples were stained with secondary antibody (Alexa Fluor 488–labeled donkey anti-rabbit IgG, 1:1000; Thermo Fisher Scientific, Waltham, USA). Last, coverslips were observed through fluorescence microscopy (Leica Microsystems, Wetzlar, Germany).

Cellular proteins were extracted using cold RIPA lysis buffer (Beyotime, Shanghai, China) containing a protease inhibitor cocktail. Proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dried milk and then incubated with primary antibodies (anti–NECTIN-1, 1:1000; Abcam) overnight at 4°C, followed by incubation with horseradish peroxidase–conjugated goat anti-rabbit IgG secondary antibody (1:1000; Cell Signaling Technology, Danvers, USA). Chemiluminescence solution (MilliporeSigma, Burlington, USA) was used to reveal the bands. Representative blots from at least three independent experiments were shown.