Mouse anti vimentin antibody

For immunocytochemistry, cells were plated on a glass‐bottom chamber slide (Matsunami Glass, SCS‐008, Osaka, Japan). After fixation, cells were washed, and permeabilized with 0.5% Triton X‐100. Subsequently, cells were washed, and incubated with blocking buffer that contained 5% goat serum and 1% BSA. After blocking, cells were incubated with the primary antibodies, mouse anti‐vimentin antibody (Sigma, V6389, St. Louis, MO, 1:20 dilution), mouse anti‐E‐cadherin antibody (BD Biosciences, 610182, San Jose, CA, 1:50 dilution), rabbit anti‐phospho‐paxillin antibody (Cell Signaling Technology, 2541, Danvers, MA, 1:20 dilution) and mouse anti‐vinculin antibody (Sigma, V9131, 1:200 dilution). Cells were washed, and stained with the secondary antibodies, rabbit anti‐mouse IgG antibody conjugated to Alexa 568 (Life Technologies, A11061, Carlsbad, CA, 1:200 dilution), goat anti‐rabbit IgG antibody conjugated to Alexa Fluor 546 (Life Technologies, A11010, 1:1000 dilution) and goat anti‐mouse IgG conjugated to Alexa Fluor 488 (Life Technologies, A11001, 1:1000 dilution). In cells immunostained for vimentin and E‐cadherin, nucleus was counterstained with DAPI. Fluorescent images were obtained using an all‐in‐one microscope (Keyence, BZ‐9000, Osaka, Japan).

The mouse anti-paxillin antibody was purchased from BD Biosciences (610052) and the corresponding secondary Alexa488 anti-mouse antibody from ThermoFisher Scientific (A-11029). The rabbit anti-fibronectin antibody was obtained from Sigma (F3648) and the corresponding secondary Alexa647 anti-rabbit antibody from ThermoFisher Scientific (A-21245). Alexa Fluor 568-coupled phalloidin was from ThermoFisher Scientific (A12380). Rat anti-E-cadherin antibody was obtained from ThermoFisher Scientific (13-1900), rabbit anti-ZO1 antibody from ThermoFisher Scientific (61-7300), mouse anti-vimentin antibody from Sigma (V2258), mouse anti-SMAD2/3 from BD Biosciences (610842), rabbit anti-p-SMAD3 from Cell Signaling (p-Ser423/425; 9520) and mouse anti-GAPDH from Sigma (G8795). For quantitative immunoblot analysis, secondary antibodies from LI-COR Biosciences, IRDye 680RD Goat anti-Mouse (926-68070) and IRDye 800CW Goat anti-Rabbit (926-32211) were used. DAPI was acquired from Sigma (D9542), recombinant human TGFβ1 protein from R & D Systems (240B-0-10), 16% paraformaldehyd (PFA) from Electron Microscopy Services (15710-S), fatty-acid free BSA from Calbiochem (126575), Trypsin/EDTA from Sigma (T4174), PBS from Gibco (14200-067), and Triton X-100 from Sigma (X-100).