Tgf β

DAN cells were grown on glass coverslips in 24-well plates overnight prior to treatment with TGF-β (5 ng/ml; Roche Diagnostics) for 72 h. Cells were treated with 1 nM Go6983 or DMSO for the last 24 h. Cells were then washed with PBS and fixed with 4% paraformaldehyde (in PBS) at room temperature (RT) for 15 min or with ice-cold methanol at room temperature (RT) for 7 min. Cells were then washed three times with PBS prior to permeabilization with 0.1% Triton X-100 in PBS for 5 min. Permeabilized cells were washed five times with PBS and blocked with blocking reagent (0.1% saponin, 10% goat serum in PBS) for 1 h at room temperature (RT). Cells were then washed once with PBS, followed by incubation with anti-E-cadherin (1 : 500) and anti-P-SMAD2/3 (1 : 200; Cell Signaling Technology) primary antibodies in blocking reagent overnight at 4°C. Cells were washed three times with PBS and incubated with labeled secondary antibodies for 1 h at RT in the dark. Cells were then washed five times with PBS and coverslips were mounted with Vectashield containing DAPI (H-1200) prior to microscopy. Images were acquired through a 20× objective with a Zeiss 710 confocal laser microscope (Zeiss, Oberkochen, Germany). Representative images are shown in all figures at the same exposure and magnification and in merged color images; co-localization is indicated by yellow and orange regions.