FIG. 3.

GSNOR negatively regulates RyR1 Ca²⁺ channel S-nitrosylation. Determination of RyR1 S-nitrosylation status using SNO-RAC assay. Ascorbic acid, which converts nitrosothiol groups to thiols, was included as a specificity control. (A) RyR1 S-nitrosylation was increased in GSNOR^−/− gastrocnemius. Upper panel: representative Western blot showing two wild-type (+/+) control (lanes 1–4) and two GSNOR^−/− gastrocnemius samples (lanes 5–8). Lower panel: representative blot showing RyR1 input. (B) Quantitation revealed a marked approximately four-fold increase in RyR1 S-nitrosylation in the gastrocnemius (gas). (C) RyR1 hypernitrosylation also occurred in GSNOR^−/− tibialis anterior muscles. (D) Gastrocnemius RyR1 protein expression was unaffected by loss of GSNOR. (E) Levels of nNOSμ and activated (Ser¹⁴⁴⁶ phosphorylated) nNOSμ were determined. Representative Western blots showing expression of nNOSμ and active ser¹⁴⁴⁶ phosphorylated nNOSμ in WT and GSNOR^−/− TA muscle. (F) Quantitative analysis showed that the fraction of active ser¹⁴⁴⁶ phosphorylated nNOSμ was unaffected by GSNOR depletion. (G) Quantitative analysis showed that nNOSμ protein expression was similar between WT and GSNOR^−/− mice. A to D, n = 8 and 7, for WT and GSNOR^−/− groups, respectively. E to G, n = 6 for all groups. * p < 0.05. SNO-RAC, S-nitrosothiol resin-assisted capture.