FIG. 4.
Cysteine 1162 is involved in the intramolecular disulfide bond essential for the activity and the targeting of DUOX2. (A) HA-DUOX2 (WT) and mutant HA-DUOX2 C1162G proteins were transiently expressed with DUOXA2 protein in HEK293 cells. The extracellular H2O2-generating activity was measured after 48 h. The expression of the WT and mutant DUOX2 was analyzed by immunoblotting in nonreducing and reducing conditions. DUOXA2 expression was assessed by using anti-DUOXA2 antibody. ***P<0.001. (B) Representative histograms of flow immunocytofluorometry experiments. Absence of cell surface expression of the mutant HA-DUOX2 C1162G was associated with an absence of cell surface expression of N-terminal cMyc epitope-tagged human DUOXA2. **P<0.01. (C) Cells were cotransfected with HA-DUOX2 or mutant HA-DUOX2 C1162G in the presence of myc-DUOXA2. Anti-myc immunoprecipitates of the cell lysates were separated by SDS-PAGE and analyzed by Western blotting. Analysis of the cell lysates used as the input in immunoprecipitation is shown in the upper two panels.