FIGURE 5.

Annexin 1 is required for p38-mediated endothelial cell migration in response to VEGF. HUVECs were co-electroporated, with siRNA to knock down human GAPDH or ANXA1 mRNAs (400 pmol of siRNA) along with pEGFP-C1 and pIRES-hrGFP-2a containing HA-tagged wild-type ANXA1 not targeted by ANXA1 siRNA. After 48 h, cells were serum-starved for 16–20 h before being treated or not treated with SB203580 (1 μm) and treated or not treated with VEGF (5 ng/ml for 4 h) Thereafter, cell migration was assayed in a modified Boyden chamber, as described under “Experimental Procedures.” The efficiency of siRNA knockdown was determined in Western blotting of the total cell extract (three right 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 = 23 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. SB, SB203580.