Phosphorylated erk1 2 thr202 tyr204

Total cellular protein extracts were obtained with radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 1% PMSF (Sigma) and 1% phosphatase inhibitor (Roche Applied Science). The concentration of protein was measured by the BCA protein assay kit (Thermo). Next, equal amounts of samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After being blocked with skimmed milk in TBST buffer (0.1 M Tris, 150 mM NaCl, and 0.1% Tween-20), the membranes were incubated overnight at 4 °C with primary antibodies against HSPB7 (Abcam, Cambridge, UK), RUNX2 (Abcam), OCN (Abcam), ERK1/2 (Cell Signaling Technology, Beverly, MA, USA), phosphorylated-ERK1/2 (Thr202/Tyr204) (Cell Signaling Technology), and GAPDH (HuaxingBio Science, Beijing, China). Subsequently, the membranes were washed three times with TBST and incubated with horseradish peroxidase (HRP) conjugated secondary antibodies for 1 h at room temperature. The bands were visualized via the ECL Western Blot Kit (CoWin Biotech). The intensity of bands was quantified using ImageJ analysis software (http://rsb.info.nih.gov/ij/) and normalized to GAPDH.