Horseradish peroxidase labeled goat anti rabbit igg h l

The total protein samples were extracted according to the kit instructions (Whole Protein Extraction Kit, Beyotime, China). The protein was separated by SDS-PAGE (voltage of 80/120 V) and transferred to PVDF membranes at a voltage of 60 V for 3 h. The membranes were blocked with 5% nonfat dry milk for 1 h at room temperature, incubated with primary antibody: GAPDH (1:8000, Cell Signaling Technology, USA); β-Actin (1:1000, Bioworld, China); OPN (1:1000, Abcam, USA); RUNX2 (1:1000, Abcam, USA); FAK (1:500, Cell Signaling Technology, USA); Talin (1:1000, Millipore, USA); Vinculin (1:800, Sigma-Aldrich, USA); Paxillin (1:1000, Becton Dickinson and Company, USA); Zyxin (1:700, Affinity, USA); YAP (1:700, Cell Signaling Technology, USA); P-YAP (1:1000, Cell Signaling Technology, USA) overnight at 4 °C with gentle shaking, and incubated with appropriate secondary antibodies: horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) (1:5000, Fdbio Science, China); horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) (1:5000, Fdbio science, China) at room temperature for 1 h. Protein expression was visualized using an exposure instrument (Tanon 5500, Tanon, China). Quantification of western blot data was performed by Gel-pro software.