FIG. 5.

ONOO⁻ increases GTPCH1 ubiquitination of intact endothelial cells. A and B: The effect of ONOO⁻ on GTPCH1 ubiquitination in cultured BAECs. Confluent BAECs were pretreated with the 26S proteasome inhibitor MG132 (0.5 μmol/L) or an equivalent volume of DMSO (control) for 30 min, and treated with ONOO⁻ (50 μmol/L) or vehicle in 0.1 mol/L HEPES buffer. BAEC lysates were incubated with ubiquitin interacting motif-agarose. GTPCH1 that was affinity precipitated with ubiquitin was analyzed by immunoblotting using anti-GTPCH1 and anti−β-actin antibodies. IB, immunoblot; Ub, ubiquitin. C and D: SIN-1 on GTPCH1 ubiquitination. BAECs were pretreated with MG132 or DMSO for 30 min and treated with 1 mmol/L SIN-1 (ONOO⁻ donor). The blot shown is representative of three independent experiments. NS, not significant. *P < 0.05 vs. control.