Regulation of membrane blebbing by ERK-1/2.

(A) Cell lysates were harvested 18 hours after the last medium change from 2 independent endothelial cultures grown either sparsely or confluent, and equal amounts of protein (40 μg) were analyzed for phosphorylated ERK-1/2 (P-ERK) and total ERK-1/2 (T-ERK). (B) Confluent endothelial cells were preincubated with either vehicle control or PD98059 (30 minutes, 25 μM) and then exposed to PDBu (200 nM) for the indicated times. Cell lysates were analyzed for P-ERK activity as in panel A. (C-H) Confluent endothelial cells grown on Permanox slides were preincubated with either PDBu (15 minutes, 200 nM; C,D) or PD98059 (30 minutes, 25 μM), followed by PDBu (15 minutes, 200 nM; E,F) or Y-27632 (30 minutes, 10 μM), followed by PDBu (15 minutes, 200 nM; G,H). Cells in panels D, F, and H were then treated with CA-4-P (30 minutes, 1 μM) and stained for actin. Bar = 40 μm.