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. 2019 Jan 8;8(1):16. doi: 10.3390/antiox8010016

Figure 4.

Figure 4

cICDH is S-nitrosylated and is denitrosylated by the GRX system. (A) 2.19 µM of recombinant wild-type cICDH or Cys-mutated versions of cICDH (cICDH-C363S) were treated with or without 1 mM GSNO for 30 min at 25 °C, and subjected to the biotin-switch assay in presence or absence of sodium ascorbate. (B) After treatment with GSNO (1 mM), the protein was treated with GRXC1 (5 µM) alone (lane 2), GRXC1, GSH (0.8 mM) and GR (0.45 µM) (Lane 3), NTRA (3 µM) alone (lane 4) and NTRA+TRXh3 (4.59 µM) (lane 5) for 30 min at 25 °C and subjected to the biotin-switch assay in the presence of sodium ascorbate. (C) The same experimental design in the presence of GSH (0.8 mM) (lane 2), GR (5 µM) (lane 3), GrxC1 (5 µM) (lane 4) and GRXC1+GR+GSH (lane 5). Afterwards, the proteins were separated by reducing SDS-PAGE and transferred onto nitrocellulose membrane. Total ICDH (bottom panel) or S-nitrosylated ICDH (top panel) was detected using an anti-His antibody.