Sc 28888

Cardiac fibroblasts/myofibroblasts were lysed with RIPA buffer and proteinase inhibitor mixture (PMSF). Nuclear and cytoplasm protein were separated by using nuclear and cytoplasm protein extraction kit (78833; Thermo Pierce, Rockford, IL, USA), according to manufacturer's instructions. Protein concentration was assessed by Bradford assay (Bio-Rad Laboratories, Shanghai, China). Total proteins were electrophoresed on 12% SDS-PAGE gels and transferred onto polyscreen PVDF transfer membranes (PerkinElmer, Boston, MA, USA). Membranes were blocked with 5% (w/v) non-fat milk in tris-buffered saline (TBS) containing 0.1% Tween 20 for 1 hr at room temperature and incubated overnight with primary anti-col3A1, anti-col1A1, anti-β-actin (sc-28888, sc-8784, sc-47778, respectively, Santa Cruz biotechnology Inc, Santa Cruz, CA, USA), anti-OPN (ab8448; Abcam) at 4°C, respectively. After washing, HRP-conjugated secondary antibody was added for 1 hr at 37°C. Detection was performed with enhanced chemiluminescence (ECL) and relevant blots were quantified by densitometry by using the accompanying computerized image analysis program.

Samples were mixed with 4× denaturing sample buffer, heated at 95°C for 10 min, and separated on a Bis‐Tris discontinuous 4–12% polyacrylamide gradient gel (NuPAGE; Invitrogen). Proteins were then transferred on nitrocellulose membranes. Membranes were blocked with 5% fat‐free milk powder in PBS‐Tween and probed for 16 h at 4°C with a primary antibody to COL3A1 (rabbit, Santa Cruz, sc‐28888) at 1:500 dilution in 5% BSA. The membranes were treated with the appropriate secondary, horseradish peroxidase (HRP)‐conjugated antibodies (Dako) at 1 : 2,000 dilution. Finally, the blots were imaged using enhanced chemiluminescence (ECL; GE Healthcare), and films were developed on a Xograph processor. The densitometry for the lanes from developed films was measured using the ImageJ software.