Fig. 5.

S-nitrosylation of GSK3β was activated by VC deprivation. (A-D) Immunohistochemistry staining, western analyses, and semi-quantification of p-Ser9-GSK3β in the livers of female Gulo^−/− pups with or without VC supplementation for 3 weeks. (E and F) Primary hepatocytes from female Gulo^−/− pups that had been deprived of VC for 3 weeks were isolated and stimulated with 60 μM VC for the times indicated, followed by western analyses. (G and H) An iodoTMT labeling strategy was used to quantify SNO-GSK3β levels in the livers of female Gulo^−/− pups with or without VC supplementation for 3 weeks. (I and J) HepG2 cells were transfected with Flag-GSK3β, then treated with 60 μM VC, 5 nM N6022, or a combination of the two. Protein samples were immunoprecipitated with a Flag antibody before being subjected to iodoTMT labeling and western analyses to detect SNO-GSK3β levels. (K–N) Western analyses and semi-quantification in L02 cells after N6022 treatment at the indicated concentrations for 30 min or at 5 nM for the indicated times. (O-R) Western analyses and semi-quantification of L02 cells treated with l-NAME at the indicated concentrations for 6 h or at 3.2 mM for the indicated times. Mean SD, n = 3, one-way ANOVA and Dunnett's multiple comparison tests; *p < 0.05, **p < 0.01 versus lane 1. Square frames define the magnified regions.