Fig. 1. Inhibition of dynamin GTPase abrogates EC survival signals. (A) BAEC transfected with AdGFP or AdK44A were incubated with vehicle or TNF-. Cells were collected 24 hours later and prepared for western blot analysis of cleaved caspase-3. Cells transfected with AdK44A showed enhanced TNF--induced BAEC apoptosis as demonstrated by a significant increase in the amount of cleaved caspase-3 from cell lysates. Blots were reprobed with V5 antibody or β-actin for verification of the expression of V5-K44A fusion protein and for protein loading control, respectively (n=3 separate experiments). (B) EC were transfected with dynamin-2 siRNA or scrambled siRNA and cells were harvested for western blot analysis after 48-72 hours or viewed by microscopy for cell morphology. (Top) Prominent morphologic changes and cell death (arrowheads) were detected in EC transfected with dynamin-2 siRNA. (Bottom) EC transfected with dynamin-2 siRNA showed increased levels of cleaved caspase-3 (n=4 separate cell preparations).