Rac1 activation contributes to the effects of CYLD on vascular endothelial cell migration and tube formation. (A) HUVECs transfected with CYLD or control siRNAs were scratched, and the level of activated (GTP-bound) Rac1 was examined by immunoblot analysis of the GST-PBD pulldown preparation with anti-Rac1 antibody. The levels of total Rac1 and tubulin were examined by immunoblot analysis of cell lysates. (B) Experiments were performed as in panel A, and the relative activity of Rac1 was measured by densitometric analysis of the blots. Data are the mean and SE from 3 experiments (*P < .05 vs control). (C) HUVECs transfected with pSUPER-CYLD and pEGFPC1-Rac1 or pEGFPC1 were scratched, and wound margins were imaged 0 and 24 hours later. Broken white lines indicate initial wound margins. Objective lens used was A-Plan 20×/0.3 NA dry (Carl Zeiss Inc). (D) Experiments were performed as in panel C, and the average migration distance of the transfected cells from the wound margin was measured. Data are the mean and SE from 2 experiments (500 transfected cells were measured for each experiment; *P < .05). (E) HUVECs transfected with CYLD or control siRNAs were plated onto matrigel, and the levels of GTP-Rac1, total Rac1, and tubulin were examined 2 hours later as in panel A. (F) HUVECs transfected with pSUPER-CYLD and pEGFPC1-Rac1 or pEGFPC1 were plated onto matrigel, and photographs were taken 25 minutes later. Objective lens used was A-Plan 20×/0.3 NA dry (Carl Zeiss Inc). (G) Experiments were performed as in panel F, and the degree of cell spreading was quantified. Data are the mean and SE from 2 experiments (600 transfected cells were measured for each experiment; **P < .01). (H) HUVECs transfected with CYLD siRNA and pEGFPC1-Rac1 or pEGFPC1 were plated onto matrigel, and the cumulative tube length was measured 4 hours later. Data are the mean and SE from 2 experiments with each performed in triplicate (*P < .05).