Fig. 2.

S-nitrosylation of recombinant ASK1 recombinant. ASK1 recombinant protein was incubated with increasing concentrations of NO-Cys for 15 min and subjected to biotin-switch assay. S-nitrosylated proteins were detected by immunoblot using an anti-biotin antibody (upper panels). Sypro ruby staining is shown as a loading control (lower panels). Controls of the biotin switch assay minus ascorbate (Asc), minus biotin, minus methyl methanethiosulfonate (MMTS) and with DTT reduction before the biotin switch are shown. (A) Representative experiment and (B) quantification of the biotin signal of three independent experiments are shown. (C) ASK1 recombinant protein was incubated with 100 µM GSNO, 100 µM GSSH and 100 µM H₂O₂ for 15 min and subjected to redox fluorescence switch assay to detect reversibly oxidized Cys. Modified proteins were detected by their fluorescent maleimide signal (upper panel). Coomassie staining is shown as a loading control (lower panel). (D) Quantification of fluorescence signal of three independent experiments. Different letters indicate a significant difference at P ≤ 0.05 (one way ANOVA, post hoc Tukey).