Phosphorylated p38mapk p p38 mapk

The expression of proteins in heart tissues was quantified as described previously [17 (link)]. Primary antibodies used from Cell Signaling Tech (Danvers, MA, USA) were phosphorylated eNOS (p-eNOS), Arg-1, Bcl-XL, p38 MAPK, phosphorylated p38MAPK (p-p38 MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated ERK1/2 (p-ERK1/2). Primary antibodies for caspase-3, Bax, and Bcl2 were obtained from GeneTex (San Antonio, TX, USA). Antibodies for superoxide dismutase 1 (SOD1), SOD2, glutathione peroxidase 2 (GPx2), AKT, p-AKT (Thr308), p-AKT (Ser473), and eNOS were obtained from Santa Cruz (CA, USA), and antibodies for $\beta$ -actin were obtained from Chemicon (Temecula, CA, USA).

Radio immunoprecipitation assay (RIPA) lysis buffer was used to extract total protein from cultured cells. Equal quantities of proteins were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Primary antibodies against RUNX2 (Abcam, Cambridge, UK), GDF5 (Abcam), phosphorylated Smad1/5/8 (pSmad1/5/8; Cell Signaling Technology, Beverly, MA, USA), p38 MAPK (Cell Signaling Technology), phosphorylated p38 MAPK (p-p38 MAPK; Cell Signaling Technology), and β-ACTIN (Abcam) were diluted 1:1000. The PVDF membranes were incubated with these primary antibodies overnight at 4 °C. After three washes with TBST, the PVDF membranes were incubated with corresponding secondary antibodies (1:10,000, Cell Signaling Technology). ImageJ software (http://rsb.info.nih.gov/ij/) was used to quantify the band intensity obtained by Western blot analysis. The signal of each target band was normalized to that of the β-ACTIN band.