Anti rabbit a0545

The expression of Collagen 1 a1 (Col1a1), Vimentin and alpha smooth muscle Actin α-SMA was evaluated by western blot. Cells were lysed with lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10% glycerol, 1% NP-40, protease inhibitor cocktail (Sigma Aldrich) and phosphatase inhibitor (Roche)), gently vortexed for 20 min at 4 °C and centrifuged for 15 min at 13,200 rpm at 4 °C. Supernatants were quantified by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Twenty micrograms of proteins from cell extracts were loaded and separated in 8% SDS-PAGE. After electrophoresis, the gels were transferred to polyvinylidene difluoride membranes (Millipore), therefore blocked (5% no fat milk in 0.1% Tween 20 TBS) and incubated with the primary Ab (1:1000 in TBST + 2% BSA; overnight at 4 °C or 2 h at room temperature): anti-α-SMA [E184] (ab32575, Abcam), anti-Vimentin (ab24525, Abcam), anti-Col1a1 (ab34710, Abcam) and anti-β-Actin (MAB1501R, Chemicon). After wash, the membranes were incubated with the appropriate HRP-conjugated secondary Ab (1:5000 in TBST + 2% BSA; 2 h at room temperature; anti-mouse A4416 and anti-rabbit A0545, Sigma). The immunoreactivity was detected by ECL reagents (Amersham), acquired with the ChemiDoc imaging system (Image Lab, Bio-Rad).