FIG. 2.

ONOO⁻ increases GTPCH1 ubiquitination. A: Purification and identification of FLAG-GTPCH1 proteins. FLAG-GTPCH1 plasmids were transfected to HEK293 for 2 days, and the proteins were analyzed by immunoblotting using anti-FLAG antibody or antibody specific for GTPCH1. B and C: Recombinant FLAG-GTPCH1 bound to anti-FLAG resin was treated with ONOO⁻ (10–100 μmol/L, separately) and incubated with Ub4. Bound proteins were detected by immunoblotting. D: Effect of zinc supplementation (zinc chloride, 10 μmol/L) on ONOO⁻-induced GTPCH1 ubiquitination. E: Effects of NO and ONOO⁻ on GTPCH1 S-nitrosylation in purified recombinant GTPCH1. CTR (−), no biotin label; CTR (+) with biotin label; vehicle (NaOH) treated and biotin labeled; ONOO⁻ (100 μmol/L), DETANO 200 μmol/L; spermine NONOate (SPERNO) 5 or 1 mmol/L, and then applied for biotin-switch assay. F: Effects of NEM on NO- or ONOO⁻-induced S-nitrosylation. The blot is representative of three to five blots from three to five individual experiments. IB, immunoblot; Ub, ubiquitin.