Expressplus 10x8

DPSC lysates (20 μg/sample) were electrophoresed on a 4–20% SDS-PAGE gel (ExpressPlus™ 10x8, GenScript Biotech Corporation, China) and transferred to nitrocellulose membranes further blocked in 5% of nonfat milk, 10 mmol/l Tris pH 7.5, 100 mM NaCl, and 0.1% Tween 20. Membranes were then probed for mouse anti-β-actin monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA) (primary antibody dilution 1 : 10,000) and mouse monoclonal anti-BSP II (Santa Cruz Biotechnology, CA, USA) (primary antibody dilution 1 : 200) and incubated in the presence of specific conjugated IgG horseradish peroxidase. Immunoreactive bands were identified using the ECL detection system (Amersham Int., Buckinghamshire, UK) and analyzed by densitometry. Densitometric values, expressed as the percentage of the integrated optical intensity ratio of BSP II and β-actin, were estimated by the ChemiDoc XRS system using the QuantiOne 1-D analysis software (Bio-Rad, Richmond, CA, USA).

Tenocytes were lysates, and 15 µg of each sample was separated on a 4–20% SDS-PAGE Gel by electrophoresis (ExpressPlus™ 10x8, GenScript Biotech Corporation, Nanjing, China). After running, the samples were transferred to nylon membranes, as already described [8 (link)]. The membranes were incubated in the presence of mouse monoclonal anti-β-actin (1:10,000) (Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti-collagen 1A1 (1:200), collagen 3A1 (1:100), mouse monoclonal anti-iNOS (1:200), mouse monoclonal anti-MMP2 (1:200) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit monoclonal anti-MMP14 (1:1000) (purchased from Abcam). After an overnight incubation at 4 °C with primary antibodies under gentle shaking, membranes were then probed with specific IgG horseradish peroxidase (HRP)-conjugated secondary antibodies and bands were identified by chemiluminescence as previously described [8 (link)]. At least three independent experiments were performed for each protein. Results are expressed as mean values ± S.D. of normalized densitometric values on β-actin.