Cellytic m kit

After 80% of confluence cells were harvested and protein content was extracted using CelLytic M kit (Sigma) according to the manufacturer’s instructions. The protein concentration was determined using Bio-Rad protein assay kit (Bio-Rad). The sample containing 25 μg of protein were solubilized Laemmli buffer and separated by 6% acrylamide with 4% acrylamide stacking gels and then transferred onto the Hybond-P PVDF membrane (GE Healthcare, UK) for 60 min at 200 mA. Then the PVDF membranes were blocked using 5% skimmed milk powder in 0.5 M Tris-buffered saline, pH 7.4 with 0.05% Tween-20 (TBST) at room temperature for 2-4 h. Immunoblot were probed with collagen type I and collagen type III primary antibody (1:5000 dilution, abcam^®#ab 34710 & abcam^®#ab7778, Korea) overnight. After three time washing with TBST the membranes were probed with HRP-conjugated anti-rabbit secondary antibody (1:2000 dilution, abcam^®# ab97051, Korea) for 60 min at room temperature. The membranes were then washed thrice with TBST for 10 mints each. Protein bands were visualized using enhanced Chemiluminescence assay kit (WesternBright^TM ECL, USA), the protein bands were scanned and quantified by Image J Software (version 1.44; Chonbuk National University). The relative density ratio of bands normalized to β-actin.