Figure 5.

Oxidative modification of the individual cysteines in SENP3 regulates p300 SUMOylation and HIF-1 transactivation. (A) The SUMOylation of p300 was detected by co-IP. HeLa cells were transfected with non-specific siRNA control or siRNA for endogenous SENP3 for 24 h, and then transfected with RGS-SENP3 WT or the mutants C243S or C532S to rescue for another 48 h. HA-SUMO3 was simultaneously co-tranfected. Cells were treated with H₂O₂ for 1 h. The proteins were co-immunoprecipitated using anti-p300 and detected by IB using the antibodies against p300 and HA. β-Actin as a control. (B) HeLa cells were transfected with non-specific siRNA control or SENP3 siRNA for 24 h, and then transfected with RGS-SENP3 WT, the mutants C243S or C532S to rescue for another 48 h. Cells were treated with H₂O₂ for 6 h. The VEGF mRNA levels were evaluated by real-time PCR and shown as folds of control. The results were shown as the Mean±SD of three independent experiments, and samples were duplicated in each experiment. Data were represented as Mean±SD. ^bP<0.05 vs siRNA SENP3 rescue WT H₂O₂0 mmol/L. ^eP<0.05 vs siRNA SENP3 rescue WT H₂O₂0.5 mmol/L. ^hP<0.05 vs siRNA SENP3 rescue C532S H₂O₂0 mmol/L.