Table 1. Hydrogen Peroxide Generating Activity (picomoles per minute per 10⁷ cells) of HEK293 Cells Expressing Nox4^a.

intact cell assay	control (a)	Nox4 (b)	b − a (%)
HEK293 cells	10.2 ± 2.5	225.9 ± 30.0	215.7 (100)
with DPI	0.85 ± 0.2	4.84 ± 1.0	4.00 (1.9)

	NADPH			NADH
cell-free assay	control (a)	Nox4 (b)	b − a (%)	control (a)	Nox4 (b)	b − a (%)
cell lysate	74.0 ± 6.3	223.5 ± 18.5	149.5 (69.3)	117.0 ± 25.1	141.4 ± 12.2	24.4 (13)
with DPI	49.2 ± 5.6	101.3 ± 15.2	52.1 (24.2)	78.5 ± 10.3	91.3 ± 7.8	12.8 (5.9)

^aIn the intact cell assay, the reaction was initiated by addition of 5 μL of cells (5−7 × 10⁴) to 95 μL of assay buffer (pH 7.5) containing 25 mM Hepes, 120 mM NaCl, 3 mM KCl, 1 mM MgCl₂, 0.032 unit of HRP, and 0.1 mM Amplex Red. The fluorescence of Amplex Red was measured for 10 min at 25 °C. In the cell-free oxidase assay, NADPH- or NADH-dependent H₂O₂ generation by cell lysates was monitored in 0.1 mL of the same buffer containing 25 μM FAD and 36 μM NADPH or 36 μM NADH. We initiated the reaction by mixing 30−50 μg of the sample/5 μL of lysate with 95 μL of the assay buffer in the presence or absence of 20 μM DPI. As a control, 5 μL of the cell lysis buffer was added to 95 μL of the reaction mixture instead of the cell lysates. The difference between the fluorescence in the presence and absence of the sample was measured, and the activity (picomoles of H₂O₂ produced per minute per 10⁷ cells or milligrams) was calculated using a linear standard curve of the fluorescence intensity vs H₂O₂ concentration (nanomolar). The cell lysates from 10⁷ cells transfected with vector alone (control) or Nox4 contained 4.53 ± 0.35 or 4.16 ± 0.30 mg of protein, respectively. Values represent the means ± the standard deviation of three experiments.