Immunoblotting of Cardiac Proteins

Immunoblotting in LV homogenates was performed to evaluate protein levels of GTPCH (1:10,000 dilution; a gift from S.Gross, Cornell University; New York), iNOS (1: 1,100 dilution; Abcam), nNOS (1:1,000 dilution; Santa Cruz Biotechnology), eNOS (1:5,000 dilution; BD Bioscience), CD102 (1:1,000; R&D systems), SERCA2A (1:5,000 dilution; Santa Cruz Biotechnology), total phospholamban (1:2,000 dilution; PLB, Badrilla), phosphor-Thr17-PLB (1:2,000 dilution; Badrilla), phosphor-Ser16-PLB (1:2,000 dilution; Badrilla), NCX1 (1:1,000 dilution, Santa Cruz), phospho-extracellular signal-regulated protein kinases (1:500 dilution; ERK1/2), total ERK1/2 (1:500 dilution;), catalase (1:5,000 dilution; Calbiochem), MnSOD (1:5,000 dilution; Stressgen Bioreagents), EcSOD (1:750 dilution; Stressgen Bioreagents), Cu/ZnSOD (1:500 dilution; Stressgen Bioreagents), and β-tubulin (1:20,000; Abcam), followed by appropriate HRP-conjugated secondary antibody (1:10,000–20,000 dilution; Promega). Protein bands were visualized by enhanced chemiluminescence (Super West Pico Chemiluminescence, Thermo Scientific).