Sc 20150

Tissue samples were homogenized in lysis buffer (50mM TrisHCl, 150mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% Triton X-100, 0.3% NP-40, 0.1 mM PMSF, and 1 µg/ml pepstatin A) and then separated by 10% SDS-PAGE under reducing conditions. After electrophoresis, samples were transferred to PVDF membranes (IPVH00010, Millipore, Bedford, MA, USA), blocked with 5% skimmed milk in TBS/0.5% v/v Tween 20 for 1 h, washed with TBS/Tween, and incubated with rabbit anti-Fibronectin (1:5000; AB2033, Millipore, MA, USA), anti-alpha smooth muscle actin (α-SMA) (1:1000; A2668, Sigma, MO, USA), anti-Collagen I (1:1000, 234167, Merck Millipore, MA, USA), anti-Arg1 (1:1000; sc-20150, Santa Cruz, Germany), anti-Arg2 (1:1000; sc-393496, Santa Cruz, Germany). Antibodies were diluted in 5% milk TBS/Tween. Blots were washed with TBS/Tween and incubated with appropriate horseradish peroxidase-conjugated secondary antibody (1:2000, Amersham, Aylesbury, UK). After washing with TBS/Tween the blots were developed with the chemiluminescence method (ECL Luminata Crescendo, WBLUR0500, Millipore, MA, USA). Blots were then probed with mouse monoclonal anti-α-tubulin antibody (1:5000, T6199, Sigma, MO, USA), and levels of expression were corrected for minor differences in loading. Quantification was expressed as arbitrary densitometric units (AU).