Protease inhibiter cocktail tablets

The yeast cells were harvested from the fermenter when the OD 600 was almost equal to 10 (at the end of exponential phase) under high and low osmotic pressure. For the extraction of the total intracellular protein, cells were centrifuged (10,000 ×g, 5 min, 4°C) and washed three times with cold Tris buffer (50 mmol/l, pH 7.4). The washed cell pellet was resuspended in 1 ml of lytic buffer (50 mmol/l Tris HCl, 150 mmol/l NaCl, 5 mmol/l EDTA, and 1 mmol/l phenylmethanesulfonyl fluoride, pH 7.4) with protease inhibiter cocktail tablets (Roche, USA), and homogenized by grinding with glass beads (0.5 mm in diameter; Sigma) in a bead beater in a fast prep cell breaker (Bio 101; level of 5.5, 4 times for 30 sec). The glass beads and unbroken cells were removed by centrifugation (13,000 rpm for 15 min). Cell extracts in the supernatant were collected into a new Eppendorf tube. The protein concentration was determined using the Bradford assay (Bio-Rad) and protein samples were stored at -80°C until analysis.

The HASMCs were collected by centrifugation at 14,000g for 10 minutes at 4 C for protein extraction using Radio-Immunoprecipitation Assay lysis buffer and one tablet/ 10 mL protease inhibiter cocktail tablets (Roche Diagnostics, Mannheim, Germany). Equal amounts of protein were loaded on an 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequently transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies (1:1000 dilution for collagen I and collagen III) overnight at 4 C. b-Actin was used as a control. Primary antibody against collagen I (catalog no. AF6220) and secondary antibody against sheep (catalog no. HAF016) were obtained from R&D Systems (Minneapolis, MN, USA). Primary antibodies against collagen III (catalog no. ab6310), bactin (catalog no. ab8226), and secondary antibody against mouse (catalog ab6789) were purchased from Abcam.