Nitrocellulose membranes

After treatment, MDCKs were lysed using RIPA buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, and 50mM Tris, pH8) supplemented with 1X protease inhibitors without EDTA (Roche). Cleared lysates collected were combined with an equal volume of 2X reducing sample buffer, heated to 95°C for 5 min, resolved on 4–12% Tris-glycine polyacrylamide gel (Thermo Fisher Scientific), and then transferred to nitrocellulose membranes containing 0.45 μm pores (Thomas Scientific). Secondary antibodies: goat anti-rabbit IgG Alexa 680 and goat anti-mouse IgG Alexa 680 (Thermo Fisher Scientific). Blots were scanned using Odyssey Scanner (LI-COR Biosciences). Western blots were quantified by normalizing the signal intensity of the E-cadherin or vimentin bands to the corresponding GAPDH signal following background subtraction.

The procedure has been described before.^{9 (link)} Briefly, protein extracts from small-intestinal tissue or cells were resuspended by homogenization in immunoprecipitation buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, and 1%Triton-X, 1X protease inhibitors). 20 μg of soluble protein were loaded on an 8% SDS-PAGE GEL, run for 90 mins at 100 V, and transferred onto nitrocellulose membranes (Thomas Scientific) at 300 mA for 90 min in a 4°C cold room. Membranes were blocked with TBS-based Odyssey Blocking buffer (LI-COR Biosciences) and incubated with primary antibodies diluted in Odyssey Blocking buffer and 0.1% Tween at 4°C overnight. After washing, the membranes were incubated with IRDye 800CW secondary antibody diluted in Odyssey Blocking buffer and 0.1% Tween at room temperature for 1 hour. Results were visualized using an Odyssey Fc Imaging System (LI-COR Biosciences) and quantified using Image Studio Software (LI-COR Biosciences).