Figure 1. PECAM-1 is required for flow-induced Rac1 activation, whereas VE-cadherin is essential for polarization of active Rac1. (A and B) Rac activation was assessed by Rac1-GTP pull-down assays in PE-RC vs. PE-KO (A) and VE-RC vs. VE-KO (B) ECs. The GTP-Rac1/total Rac1 ratio was quantified using ImageJ (National Institutes of Health). (C and D) ECs plated on fibronectin were transiently transfected with EGFP-Rac and mCherry-PBD. 24 h after transfection, cells were subjected to shear stress or kept as static control. FRET images were obtained using MetaMorph software (MDS Analytical Technologies) to show spatial Rac1 activation. Cells were first scored for total FRET signal. Cells showing a positive FRET signal were then scored for FRET directionality. Values are mean ± SEM, n = 3 and >100 cells were scored per condition. *, P < 0.05; **, P < 0.01. Bar, 10 µm.
Figure 1.

PECAM-1 is required for flow-induced Rac1 activation, whereas VE-cadherin is essential for polarization of active Rac1. (A and B) Rac activation was assessed by Rac1-GTP pull-down assays in PE-RC vs. PE-KO (A) and VE-RC vs. VE-KO (B) ECs. The GTP-Rac1/total Rac1 ratio was quantified using ImageJ (National Institutes of Health). (C and D) ECs plated on fibronectin were transiently transfected with EGFP-Rac and mCherry-PBD. 24 h after transfection, cells were subjected to shear stress or kept as static control. FRET images were obtained using MetaMorph software (MDS Analytical Technologies) to show spatial Rac1 activation. Cells were first scored for total FRET signal. Cells showing a positive FRET signal were then scored for FRET directionality. Values are mean ± SEM, n = 3 and >100 cells were scored per condition. *, P < 0.05; **, P < 0.01. Bar, 10 µm.

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