FIGURE 5.

Superoxide production by NOX5 requires H_V1. A, superoxide production by K562/NOX5 cells preincubated for 5 min with or without 1 mm ZnCl₂ and then stimulated with ionomycin (1 μm), PMA (1 μg/ml), or H₂O₂ (100 μm) was measured and expressed as described in the legend to Fig. 2. The data expressed are the mean ± S.E. of 6 independent experiments. Number signs indicate statistical significance versus stimulated cells in the absence of ZnCl₂. ###, p < 0.001. B, K562/NOX5 cells were transiently transfected with scrambled (scr) siRNA or H_V1-specific siRNA and tested for superoxide production stimulated by H₂O₂ (100 μm), ionomycin (1 μm), or PMA (1 μg/ml). Immunoblot analysis using H_V1 antibody (above) was performed to ensure that the down-regulation of H_V1 protein by siRNA was effective. Actin antibody was used to control for protein loading. The data expressed are the mean ± S.E. of 5 independent experiments. The number signs indicate statistical significance versus stimulated cells transfected with scrambled siRNA. ##, p < 0.01; ###, p < 0.001. C, immunoprecipitation of NOX5 or H_V1 protein was performed using total protein lysates isolated from K562 cells or K562/NOX5 cells. The content of the immunoprecipitates was analyzed by immunoblot using H_V1 and NOX5 antibodies. The results are representative of 3–5 experiments. D, families of proton currents were recorded during pulses in 10-mV increments up to +60 mV in K562 (lower tracings) and K562/NOX5 cells (upper tracings) before and then after stimulation with PMA (60 nm), and finally after addition of GFX (3 μm).