Enhanced chemiluminescence kit

Total protein was extracted from the treated cells with the help of RIPA buffer (Beyotime, China). After examination of protein concentration, each sample (20 μg) was isolated by SDS-PAGE and electroblotted onto a PVDF membrane. After blocking with 5% nonfat milk for 1 h, the membranes were incubated with primary antibodies against Cortactin (ab33333, abcam, 1:1000), MMP2 (10373-2-AP, Proteintech, 1:1000) MMP9 (10375-2-AP, Proteintech, 1:1000), ERK (9102, CST, 1:1000), PCNA (sc-56, SantaCruz, 1:1000), Ki67 (ab15580, abcam, 1:1000), caspase 3 (9662, CST, 1:1000), and GAPDH (60004-1-Ig, Proteintech, 1:5000) overnight at 4 °C. Next, the membranes were incubated with the secondary antibody at ambient temperature for 2 h. Finally, an enhanced chemiluminescence kit (Invitrogen, USA) was applied to develop the signals, and Image J software was used to analyze the grey values of target bands.

Ligament fibroblasts were lysed using RIPA lysate (Beyotime Institute of Biotechnology) at 4°C for 30 min. The supernatants were collected via centrifugation at 7,200 × g at 4°C for 10 min. Total protein was quantified using the bicinchoninic acid assay kit (Beyotime Institute of Biotechnology) and 60 µg protein/lane was separated via 10% separating gum and 5% concentrating gum. The separated proteins were subsequently transferred onto polyvinylidene difluoride membranes and blocked with 5% skim milk for 1 h at 37°C. The membranes were incubated with primary antibodies against: Notch2 (cat. no. ab8926), runt-related transcription factor 2 (RUNX2; cat. no. ab23981), osteocalcin (cat. no. ab93876), GAPDH (cat. no. ab9485) and rabbit anti-human (all 1:5,000 and from Abcam) overnight at 4°C. Following the primary incubation, membranes were incubated with horseradish peroxidase-labeled goat-anti-rabbit IgG secondary antibody (1:5,000; ca. no. ab6721; Abcam) for 1 h at 25°C. The protein blots were visualized using an enhanced chemiluminescence kit (Invitrogen; Thermo Fisher Scientific, Inc.). Protein bands were assessed using a luminescent image analysis software (Quantity One 1-D Analysis software; version 4.6.9; Bio-Rad Laboratories, Inc.). GAPDH was used as the internal control.

For Western blotting, tissue lysates were obtained using radioimmunoprecipitation assay lysis buffer (Solarbio). Thirty micrograms of protein were added to the lane for polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking, the blotted membranes were incubated with the following primary antibodies at 4°C overnight: PPAR-γ (1:1000; Abcam), C/EBP-α (1:1000; Abcam), and β-actin (1:1000; Abcam) monoclonal antibodies. The blots were then incubated with the corresponding secondary antibodies (1:5000; Abcam) at room temperature for 1 hour. The results were checked using an enhanced chemiluminescence kit (Invitrogen) and analyzed with PC densitometry software (Bio-Rad Laboratories, Hercules, CA).