Fig. 2. Endogenous and exogenous NO promotes survival signals that counterbalance TNF--induced EC apoptosis. (A) BAEC transfected with scrambled siRNA or eNOS-siRNA were incubated with TNF- and/or VEGF for 24 hours. Cells were harvested for western blot using antibodies for cleaved caspase-3. VEGF protected cells from TNF--induced apoptosis as shown by the reduced level of cleaved caspase-3 from cell lysates. However, VEGF protection was completely abrogated in cells transfected with eNOS-siRNA. The blot was reprobed with β-actin for a control of protein loading, and for eNOS to confirm siRNA knockdown. (B) BAEC were treated with SNP or TNF- or both and collected for protein extraction and western blot. TNF- treatment of cells resulted in increased levels of activated caspase-3. Levels of activated caspase-3 were diminished in cells treated with SNP and TNF- when compared with cells treated with TNF- alone. A representative panel of western blots of triplicate experiments is shown in the top panel, with densitometric analysis of pooled experiments shown in the bottom panel.