FIG. 3.

The ubiquitination of Prx1 in OGD-treated HBMEC. (A) The time-dependent change of OGD-induced Prx1 and ubiquitin expression in HBMECs was detected by Western blot. (B) Densitometry of the Western blots for (A) was normalized by the level of β-actin as an internal control. (C) OGD-treated HBMEC endothelial cells were lysed and subjected to immunoprecipitation with antibodies to ubiquitin. The resultant precipitates were then subjected to immunoblot analysis with antibodies to Prx1. (D) Immunoprecipitation of Prx1 from lysed HBMEC with or without OGD treatment were collected for immunoblot analysis with ubiquitin antibody. (E) The ubiquitin-K48R mutant transfection attenuates Prx1 ubiquitination in OGD-treated endothelial cells. HBMEC were cultured and transfected with plasmid DNA encoding the ubiquitin-K48R mutant or an empty plasmid. (F) Densitometry of the Western blots for (E) was normalized by the level of β-actin as an internal control. Immunoblots are representative of three independent experiments. Data are expressed as the percentage of values of control (mean±SEM). *p<0.05; **p<0.01 versus control; ^##p<0.01 versus OGD. HBMEC, human brain microvascular endothelial cell.