Figure 3

3H-naloxone binding is increased in the presence of the gp91 ^phox A. Neutrophils (WT and gp91^phox-/-) whole cell and cell membrane, B. COS-7 (WT: transfected with empty vector; gp91^phox-p22^phox: transfected with gp91^phox-p22^phox subunit and NOX2: transfected with gp91^phox-p22^phox and cytosolic subunits) and HEK293 (WT and gp91^phox-p22^phox transfected with gp91^phox-p22^phox, detailed transfection results are shown in Figure 4) were incubated with ³H-naloxone (6 nM) in binding buffer (HBSS + 10% serum) for 2 hours at 4°C. Afterward, the buffer was removed and cells or membrane were washed seven times with ice-cold HBSS. C. Plasma membranes isolated from COS7 and COS7-gp91^phox-p22^phox were used to perform the radiolabeled binding directly or to conduct IP using anti-gp91^phox antibody and the radio-labeled binding assay (IP-binding assay). Then, cell or membrane was mixed with 10 ml of Ultima Gold scintillation fluid and counted for radioactivity. Results are expressed as a percentage of the control and are mean ± SEM of three independent experiments performed in duplicate. **P < 0.01 compared with corresponding WT group. IP, immunoprecipitation; HBSS, hank's buffered salt solution.