Bcl 2 associated x protein

Homogenization of flash‐frozen tissue from the ischemic area of the left ventricle was used to make lysates, from which protein concentration was determined using a radioimmunoprecipitation assay (Pierce BCA Protein Assay Kit). Western blot analysis was done using whole‐tissue lysates fractionated onto 4% to 15% Tris‐glycine Bio‐Rad gels (Life Science Research, Hercules, CA) and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). Blocking buffer (Thermo Fisher Scientific) was applied for 1 hour before incubation with primary antibodies. Primary antibodies (against endothelial NO synthase [eNOS], phosphorylated eNOS [serine 1177], B‐cell lymphoma‐2 [BCL‐2], BCL‐2–associated death promoter, BCL‐2–associated X protein, transforming growth factor‐β, and vascular endothelial growth factor receptor 2, all from Cell Signaling, Danvers, MA) were applied at a 1:1000 dilution and incubated overnight at 4°C. Loading error was corrected using anti‐GAPDH on all membranes. A secondary anti‐rabbit antibody (Cell Signaling) was added at room temperature for 1 hour. Images were visualized using a digital camera to capture enhanced chemiluminescence images (G‐box; Syngene, Cambridge, England). Band densitometry was determined as arbitrary light units using Image J software. After normalization to GAPDH, data are presented as fold change compared with control±SEM.