S-nitrosylation of PDI in astrocytes following OGD/reperfusion.
A. In the presence of both ascorbate and biotin-HPDP, astrocytes following OGD 8 h/reperfusion 24 h treatment had detectable SNO-PDI, indicating the specificity of biotin-switch assay. B. PDI was S-nitrosylated in cultured astrocytes following OGD/reperfusion. Densitometric quantitation showed significant differences of SNO-PDI levels between the control group and the OGD/reperfusion groups. C. 1400W significantly inhibited NO production; the iNOS protein expression remained unaltered in cultured astrocytes following OGD 8 h/reperfusion 24 h. The cultured astrocytes were pretreated with various concentrations of iNOS inhibitor 1400W, which suppressed the S-nitrosylated PDI formation under OGD 8 h/reperfusion 24 h. The level of SNO-PDI was reduced with the increased concentration of 1400W (1, 10, and 50 μM). Moreover, 1400W suppressed S-nitrosylation of PDI, with the maximum effect seen at the concentration of 50 μM. Three independent experiments were performed. Data were presented as mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA (analysis of variance) with Tukey’s post-hoc test.