Western Blot Analysis of NECTIN4 and Signaling

Western blotting was performed as reported previously [46 (link)]. The antibodies used were as follows: rabbit anti-human NECTIN4 (1:1000, Abcam; ab192033), rabbit anti-human Akt (1:1000, #9272), rabbit anti-human phospho-Akt (1:2000, #4060), rabbit anti-human ERK (1:1000, #9102), rabbit anti-human phospho-ERK (1:2000, #4370), rabbit anti-human MEK (1:1000, #9122), rabbit anti-human phospho-MEK (1:1000, #9154), rabbit anti-human EGFR (1:1000, #4267), rabbit anti-human β-actin (1:2000, #4970), and goat anti-rabbit IgG horseradish peroxidase-linked secondary antibody (1:10,000, #7074) (all from Cell Signaling Technologies, Danvers, MA, USA). Immunological bands were visualized with SuperSignal^TM West Pico Chemiluminescence Substrate (Thermo Fisher Scientific; 34580) and captured with the ChemiDoc^TM XRS Plus System (Bio-Rad Laboratories; 1708265J1PC). The signals of bands were measured with Image Lab Software (Bio-Rad Laboratories).