Regulation of TSP1 and Id1 by MEK/ERK5. (A) Endothelial cell chemotaxis to VEGF (100 pg/mL) or control (bovine serum albumin) using a Boyden chamber assay after treatment with U0126 (10μM) or PD98059 (10μM or 30μM). Bars represent mean ± SD (n = 4). ***P < .001. (B) TSP1 and Id1 protein expression after expression of CA or DN MEK5 in HMVECs via lentiviral transduction. After Western blot analysis (top), quantitation was performed using LI-COR software analysis for TSP1 (□) and Id1 () normalized to α-tubulin expression (bottom). Data plotted are averages of 3 independent experiments ± SE. (C) Analysis of TSP1 protein expression in HMVECs expressing CA MEK5 or control after treatment with 8CPT. After Western blot analysis (top), quantitation was performed, as above (bottom). Data shown are representative of 2 independent experiments. (D) Detection of pERK5 in HMVECs treated with VEGF (10 ng/mL) for 5 minutes or pretreated with 8CPT (100μM) for 10 minutes, followed by VEGF treatment. Samples were analyzed by Western blotting. Data shown are representative of 2 independent experiments.