Goat anti rabbit igg horseradish peroxidase linked secondary antibody

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).

NRK-52E cells were lysed in a lysis buffer (150 mM NaCl, 1 mM EDTA, 50 mM Tris, 1% NP-40, 1% Triton X-100, and 1% protease inhibitor cocktail (Sigma-Aldrich, Kyoto, Japan)) and centrifuged to collect the supernatant. The loading samples containing "1" to "5" μg of protein were electrophoresed on SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking the PVDF membranes with 5% nonfat milk in TBST (Tris-buffered saline (TBS) containing 0.1% Tween 20) for 1 h, the membranes were incubated with E-cadherin, α-SMA or GAPDH, diluted up to 1:2000, at 4°C overnight. Goat anti-rabbit IgG, horseradish peroxidase-linked secondary antibody (Cell Signaling Technology), diluted up to 1:2000 was detected by adding enhanced chemiluminescent reagent. Blots were stripped with Restore^™ Western Stripping Buffer (Thermo Fisher Scientific) and re-probed with different antibodies. The band intensity was quantified from scanned membrane images using the ImageJ Fiji software.