Fig. 5

D609 inhibits expression of human and mouse type II NOS protein

D609 (50 µg/ml) was added to cell RAW 264.7 or human A549 cell cultures 30 min before they were activated as described for Fig. 1. Following incubation for 24 hr, cells were washed with PBS and lysed in 2% SDS; the protein concentration was determined with a DC Protein Assay kit (Bio-Rad). An aliquot of 50 µg of each sample was electrophoresed through a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The presence of a 130-kDa type II NOS band was detected with rabbit anti-type II NOS antibodies (Santa Cruz Biotechnology, Inc.). For both human (hiNOS) and mouse (miNOS) cell lysates, unactivated (lane 1) cells were compared with activated (lane 2), and activated, D609-treated cells (lane 3).