Effects of calphostin C on H₂O₂-promoted eNOS phosphorylation.

<p>In panel A, cardiac myocytes were incubated with nifedipine (100 µM, 30 min) or vehicle, then treated with H₂O₂ (25 µM) and analyzed in immunoblots probed with phospho-protein kinase C (PKC) phosphorylation (βII Ser660) or GAPDH antibodies. Panel B shows a representative experiment looking at the effects of calphostin C on H₂O₂-promoted eNOS phosphorylation. Freshly isolated adult murine cardiac myocytes were treated with calphostin C (1 µM, 30 min) or vehicle before treatment with H₂O₂ (25 µM, 15 min). Cell lysates were resolved by SDS-PAGE and probed using antibodies directed against phospho-eNOS Ser1177, phospho-eNOS Ser633, total eNOS, phospho-PKC, or GAPDH. Densitometric analyses from pooled data, plotting the fold increase of the degree of protein phosphorylation (in arbitrary units) relative to the signals present in unstimulated cardiac myocytes are also shown in this figure. Each data point represents the mean ± S.E. derived from three independent experiments, *indicates p<0.05 for respective phospho-protein versus unstimulated cells (<i>ANOVA</i>).