Peroxidase coupled anti goat igg

After tissues were homogenized in liquid nitrogen, the homogenate was lysed on ice for 30 min in lysis buffer (BioTeKe, Beijing, China). The lysates (20–40 μg) of total protein were loaded per well and separated on a 10% SDS-polyacrylamide gel. Primary antibodies were anti-CTGF (1:5000 dilution, Abcam, Shanghai, China), anti-collagen-II (1:5000 dilution, Abcam), anti-collagen-IV (1:5000 dilution, Abcam), anti-Smad 2/3 (1:1000 dilution, Cell Signaling Technology, Boston, MA), anti-Smad 7 (1:1000 dilution, Santa Cruz Biotech, Santa Cruz, CA), anti-p-Smad2 (1:1000 dilution, Cell Signaling Technology), and anti-actin antibodies (1:5000 dilution, Santa Cruz Biotech). The secondary antibody was a peroxidase-coupled anti-goat IgG (GE Healthcare). The membrane was exposed to ECL Hyperfilm (GE Healthcare), and the film was developed. The bands were quantified by densitometry using ImageJ. Results were from triplicate experiments.

After treatments, cells were lysed on ice for 30 min in lysis buffer (containing 0.15 M NaCl, 30 mM Tris, 1 mM phenylmethanesulfonyl fluoride, 1% Triton X-100, 1 mM EDTA, 10 μg/ml leupetin, 2 μg/ml pepstatin, 2 μg/ml aprotinin and 2 mM Na₃VO₄). A total of 20–40 μg of protein was separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Primary antibodies were: anti-TIMP-1, anti-procollagen-I, anti-procollagen-III and anti-β-actin (Sigma). The secondary antibody was a peroxidase-coupled anti-goat IgG (GE Healthcare). The membrane was exposed to ECL Hyperfilm (GE Healthcare), and the film was developed. The bands were quantified by densitometry using Image J. Results were obtained from triplicate experiments.