Figure 12. Pretreatment with L-NAME does not affect peptide inhibition of apoptosis.

HUVEC were grown to 70–80% confluence in a 96-well tissue culture plate. Media was removed from the wells and to each of quadruplicate wells was added 200 µl of either fresh media (untreated) or fresh media containing 2 mM L-NAME and the cells incubated 5 h at 37°C. To each well was then added the indicated peptide to achieve a concentration of 50 µM and the cells incubated an additional 3 h at 37°C before addition of staurosporine to a final concentration of 500 nM. The plate was incubated ON at 37°C and the cells processed for measurement of apoptosis using the Cell Death Detection Assay (Roche Diagnostics) per the manufacturer's instructions.