Fig. 3: S-nitrosylation of Cys337 in ERO1 enhance ERO1 activity.
a The MS/MS spectrum of S-nitrosylated peptide containing Cys337 in ERO1. b The MS/MS spectrum of S-nitrosylated peptide containing Cys336 in ERO2. c The relative intensity of S-nitrosylated peptides containing Cys337 in ERO1, Cys336 in ERO2, and Cys346 in PDI7, in wild type and hot5-4 mutant. The S-nitrosylated Cys residues are highlighted in red. Error bars, SD (n = 3 biological replicates). * p < 0.05, ** p < 0.01, two-tailed unpaired t test. d Analysis of S-nitrosylation of recombinant wild-type and Cys337Ser mutated ERO1 without or with GSNO treatment. The S-nitrosylated recombinant HIS-ERO1 was detected by anti-biotin immunoblot and the loading of HIS-ERO1 and HIS-ERO1C337S was measured by anti-HIS immunoblot, respectively. Images shown are representative of at least two independent experiments. e Analysis of ERO1 and ERO1C337S activities without or with GSNO treatment by gel-based RNase A refolding assay. Images shown are representative of at least three independent experiments. f The reduced SDS-PAGE result show the amount of wild-type and Cys337Ser mutated recombinant ERO1 proteins used in (e). Images shown are representative of at least three independent experiments. g The control reactions without AtERO1. RNase A was separated by 15% nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie Blue staining. Dred: denatured and reduced RNase A; Pox: partially oxidized RNase A; Fox: fully oxidation RNase A. Images shown are representative of at least two independent experiments.
