CD11bϩ cells were either co-cultured with d7EB cells as described above, exposed to DETA NO, or cultured alone. After stimulation with LPS, 1 g/ml, for 12 hours, the cells were lifted by gentle scraping. Human cells, which do not express CD11b, were removed via positive selection using CD11b magnetic beads (Miltenyi Biotek, Inc.). Live CD11bϩ cells were lysed in 1ϫ radioimmunoprecipitation assay lysis buffer containing protease inhibitor cocktail, 60 L/10 ml PBS (Sigma-Aldrich Corp.). Lysates were centrifuged at 14,000 rpm for 10 minutes at 4°C. Supernatants were collected, and the protein concentration of each sample was measured using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Chemical Co., Rockford, IL). For each sample, 50 g of protein was loaded onto lanes of Nu-PAGE 4% to 12% Bis-Tris gel (Invitrogen Corp.). Proteins were transferred to nitrocellulose membranes (Millipore Corp.). After incubation in blocking solution (5% dry milk in Tris-buffered saline solution with Tween 20) at room temperature for 1 hour, membranes were immunoblotted (24 hours at 4°C) with anti-iNOS rabbit polyclonal antibody (ab3523) and anti-eNOS mouse monoclonal antibody (ab76199) (both from Abcam Inc., Cambridge, MA), followed by secondary horseradish peroxidaseconjugated goat anti-rabbit or mouse (1:1000). Peroxidase labeling was detected using the ECL Western Blotting Detection System (GE Healthcare, Piscataway, NJ).