Vimentin vim

After 24 h of treatment, cells were lysed in a buffer (50 mM Tris–HCl pH 5.5, 150 mM NaCl, Triton X-100 0.5%) added with a complete protease inhibitor (Roche Applied Science). Equal amounts of proteins were denatured at 100 °C for 10 min and subjected to SDS-PAGE on 10% polyacrylamide gel. After the run, the proteins were electro-transferred on nitrocellulose membranes. Saturation of non-specific sites was performed for 2 h at room temperature in 5% milk in the TBST buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20). The membranes were exposed to primary antibodies directed against GAPDH (Santa Cruz sc-25778), α-SMA (Sigma A5228), VIMENTIN (VIM) (Santa Cruz 7557), FIBRONECTIN (FN) (Santa Cruz sc-9068) overnight at 4 °C and subsequently incubated with a secondary antibody conjugated with peroxidase for 1 h at room temperature. The signal was detected with Luminata™ Forte Western HRP Substrate (Millipore) according to the manufacturer’s instructions and the signal was acquired with Mini HD9 (UVItec, Cambridge). The band intensities were quantified using the UVItec Image Program.