Proper spatial activation of Rac1 is required for flow-induced ROS production. (A) ECs were transiently transfected with wild-type Rac (RacWT) or constitutively active Rac (RacV12) constructs. 24 h after transfection, ECs were loaded with 2,7-dichlorodihydrofluorescein diacetate (H₂-DCFDA) at 37°C for 30 min before the onset of flow shear stress was applied to the cells and ROS production was assessed by measuring the fluorescence of the cell lysates at excitation 485 nm/emission 535 nm. (B) VE-RC or VE-KO cells grown to confluence were loaded with H₂-DCFDAat 37°C for 30 min before the onset of flow. Shear stress was subsequently applied in continued presence of the H₂-DCFDA dye for the indicated times. ROS production was assessed by measuring the fluorescence of the cell lysates at excitation 485 nm/emission 535 nm.