Fig. 5.

ERK potentiates PMA-dependent Nox5 activity in both intact cells and isolated activity assays but does not modify intracellular calcium levels. A: COS-7 cells were cotransfected with HA-Nox5 and either control (lacZ), WT-ERK2, or DN-ERK2 (K52R), and superoxide release was measured in response to PMA (100 nM). B: superoxide release from Nox5 in an isolated activity assay. Nox5 was extracted from detergent resistant microdomains of cells cotransfected with HA-Nox5 and a control (LacZ) or WT-ERK2 and exposed to PMA. RFP, red fluorescent protein. Nox5 was incubated in a buffer containing 100 nM CaCl₂, 100 μM FAD, and superoxide, initiated by injection of 100 μM NADPH (indicated by arrow). C and D: measurement of intracellular calcium in aequorin-transfected COS-7 cells in response to ionomycin (1 μM; C, initiation indicated by arrow) and PMA (100 nM; D, initiation indicated by arrow) over the times indicated. Results are presented as means ± SE; n = 4–6 experiments. *P < 0.05 vs. lacZ control.