Ecl kit

Approximately 1 × 10⁷ cells were solubilized in lysis buffer purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) was utilized to separate the proteins. Afterwards, approximately 60 µg of the purified proteins was transferred to a polyvinylidene difluoride (PVDF) membrane. Then, the membrane with the adsorbed proteins was incubated with Tris‐buffered saline with Tween 20 (TBST) buffer obtained from Fanke Biotech Co., Ltd. (Shanghai, China) at room temperature, supplemented with 5% non‐fat milk. After 1 hour, the membrane was incubated with the primary antibodies overnight at room temperature, followed by incubation with the corresponding secondary antibody for 4 hour. In the present study, the primary antibodies used were as follows: rabbit anti‐α‐SMA (ab124964, 1:10 000 dilution; Abcam), rabbit anti‐ITGB3 (ab218435, 1:5000 dilution), rabbit anti‐PARP‐1 (1:1000; Cell Signaling) and γH2AX (1:250; Cell Signaling). The secondary antibody was goat anti‐rabbit IgG (1:5000 dilution; Beyotime) labelled with horseradish peroxidase (HRP). An electrochemiluminescence (ECL) kit and ImageJ software from Media Cybernetics (Rockville, MD, USA) were used to determine the chemiluminescent and relative protein expression, which was presented as the density ratio compared to GAPDH.

Approximately 1 × 10⁷ cells were solubilized in lysis buffer purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Twelve percent SDS-PAGE was utilized to separate the proteins. Afterwards, approximately 60 μg of protein was transferred to a polyvinylidene difluoride (PVDF) membrane. Then, the membrane adsorbing the proteins was incubated with TBST buffer (Tween 20) at room temperature containing 5% nonfat milk. After 3 h, the membrane was incubated with primary antibodies for 3 h at room temperature. After washing with TBST buffer (Tween 20), the membranes were treated with a matched secondary antibody for 1 h. The following primary antibodies were used: rabbit anti-SMAD3 (1:5000 dilution, ab40854) and rabbit anti-PAX6 (1:1000 dilution, ab5790); the secondary antibody was goat anti-rabbit labeled with HRP (horseradish peroxidase) (1:5000 dilution, ab205718). All antibodies were obtained from Abcam (Cambridge, MA, USA). An ECL kit and the Image-Pro plus software, version 6.0, from Media Cybernetics (Rockville, MD, USA) were used to determine the chemiluminescent and relative protein expression, respectively, which was represented as the density ratio vs. GAPDH.