Figure 4
S-nitrosylation of ER stress sensor proteins.
(a) SH-SY5Y neural cells transduced with vectors expressing wild-type IRE1α, PERK, or ATF6 were incubated with physiological NO donors (SNOC or GSNO) to determine S-nitrosylation. Control cells were exposed to old SNOC, from which NO had been dissipated, or glutathione. S-Nitrosylated proteins were detected by performing biotin-switch assays. (b) Top: SH-SY5Y neural cells, transduced with wild-type (WT) or C-to-S HA-tagged IRE1α mutants, were exposed to 100 μM SNOC or control for 1 h. SNO–IRE1α was detected by biotin-switch assay with anti-HA antibody. Bottom: Total IRE1α. (c) Quantification of XBP1 mRNA splicing is shown from (b). Values are mean ± s.e.m. (n = 4; P < 0.05 or 0.01 by ANOVA). (d) XBP1 mRNA splicing in IRE1α-null MEFs. IRE1α-null MEFs transduced with WT or IRE1α C-to-S mutants were exposed to 100 μM SNOC for 12 h and XBP1 mRNA splicing was assessed by performing RT-PCR. (e) Quantification of XBP1 mRNA splicing. Values are mean ± s.e.m. (n = 4; P < 0.05 by ANOVA). (f) Overexpression of IRE1α(C931S) mutant or spliced XBP1 mRNA prevented NO-induced cell death of IRE1α-null MEFs. Cell viability was estimated by performing WST-1 assay (see Methods). Values are mean ± s.e.m. (n = 4; P < 0.05 by ANOVA).
