Fig. 4.

Overexpression of Glrx-1 preserves Rac1 activity in endothelial cells under conditions of metabolic stress. A–B: Effect of HPHG treatment on the activity of RhoA, Rac1, and Cdc42 in HAECs. HAECs were exposed to HPHG at the indicated concentrations for 2 h, followed by activity assays for RhoA, Rac1, and Cdc42. A: Representative Western blotting result for RhoA, Rac1, and Cdc42 in the GTP-bound form and 10% of total input. B: Bar graph shows the corresponding densitometric analysis of active RhoGTPases (ratio of GTP-bound form over total GTPases) from three independent experiments. *p<0.05 vs. vehicle group. C–F: Effect of PrS-SG induction on the activity of Rac1 and RhoA in HAECs. HAECs exposed to 50 μM diamide (C and E, left panel), or 20 μg cell lysates incubated with a mixture of 1 mM GSSG/3 mM GSH on ice for 30 mins (C and E, right panel), were subject to RhoA and Rac1 pull-down activity assays. D and F: The corresponding Densitometric analysis of blot data for the activity of Rac1 and RhoA, respectively. *p<0.05 vs. vehicle group. G–H: Effect of Glrx-1 overexpression on Rac1 activity in HAECs exposed to HPHG. HAECs were infected with an adenovirus expressing hGlrx-1 or a control adenovirus (AdLacZ) for 24 h, followed by HPHG treatment (200 μM palmitate-BSA conjugate, 25 mM glucose) for 2 h. Cells were then subject to Rac1 activity assay. *p<0.05 between indicated group.