FIGURE 6.

LIMK1 is required for p38-mediated endothelial cell migration in response to VEGF. HUVECs were co-electroporated, with siRNA to knock down human GAPDH or LIMK1 mRNAs (200 pmol of siRNA) along with pEGFP-C1. After 48 h, cells were serum-starved for 16–20 h before being treated or not treated with VEGF (5 ng/ml for 4 h) and being assayed for migration in a modified Boyden chamber, as described under “Experimental Procedures.” The efficiency of siRNA knockdown was determined in Western blotting using the total cell extracts (lower two panels).The results of cell migration are expressed as the ratio of the number of fluorescent endothelial cells that have crossed the membrane in each experimental condition over the number of fluorescent cells that have crossed the membrane in untreated conditions (mean number = 12 cells). Data points represent the mean ratio with 95% confidence intervals. p was determined by Student's t test. Representative results of three separate experiments are shown.