Figure 2.

Erk mediates effects on permeability downstream of PAK. (A) BAECs were pretreated with 20 μg/ml PAK N-terminal (NT) peptide that blocks Nck binding; a mutated, control peptide; or the MEK inhibitor U0126 (25 μM). After 1 h, 25 ng/ml VEGF was added for 30 min. Cells were then fixed and stained for activated Erk. (B) Cells as described in A were extracted and analyzed for Erk phosphorylation by Western blotting. Quantitation: values are means ± SE for phospho-Erk normalized total Erk (n = 3). The increase after VEGF in the control sample is statistically significant, as are the differences between VEGF-induced permeability in control versus PAK peptide, and between control and UO126 (p < 0.01 in all cases). (C) BAECs on 3-μm filters were transfected with dominant-negative MEK1 (DN MEK), pretreated for 1 h with 20 μg/ml PAK N-terminal peptide or mutated, control peptide, or pretreated with 25 μM U0126. Cells were left untreated or stimulated for 1 h with 25 ng/ml VEGF, 50 ng/ml bFGF, or 10 μM histamine. Leakage of HRP across the filter was assayed as described in Materials and Methods. Values are means ± SD, n = 3.