W., Dodia C., Feinstein S. USA). MPMVECs were obtained from the Insitute for Environmental Medicine Cell Culture Core and maintained in culture, as described previously (15); these cells had been isolated from C57 BL/6 mice and were used at passage 4. We used the following antibodies: mouse monoclonal anti-p67phox (BD Biosciences San Jose, CA, USA), rabbit polyclonal anti-p47phox (Upstate); mouse monoclonal anti-IgG and monoclonal anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA); polyclonal donkey anti-mouse IR-680 and goat anti-rabbit IR-800Dye secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA); and mouse monoclonal anti-phosphothreonine and rabbit polyclonal anti-phosphoserine (Zymed, South San Francisco, CA, USA). Rabbit polyclonal antibodies to His-tagged human Prdx6, mouse Prdx6, and phosphorylated Prdx6 have been described previously (9, 16). An antibody that reacted specifically to nonphosphorylated Prdx6 was generated by 21st Century Biochemicals (Marlboro, MA, USA); rabbits were immunized with a peptide, TKPVATPVDWKK, conjugated with an N-terminal cys-aminohexanoic acid moiety and a second construct with a C-terminal cys preceded by an aminohexanoic acid spacer. The immune serum, after repeated depletion by incubation with peptide one containing phosphothreonine replacing threonine at position 6, recognized Prdx6 but Indacaterol not phosphoPrdx6 on Western blot analysis (Fig. 1independent experiments. Statistical significance was assessed with SigmaStat software (Jandel Scientific, San Jose, CA, USA). Group differences were evaluated by 1-way ANOVA followed by Student’s test as appropriate. Differences between mean values were considered statistically significant at 0.05. RESULTS Interaction of Prdx6 with p67phox in cells Coimmunoprecipitation of p67phox and Prdx6 from MPMVECs was used to analyze the interaction of the two proteins. As described previously for PMNs (14), anti-p67phox antibody coimmunoprecipitated Prdx6 and p67phox (Fig. 1with minimal fluorescence in phosphorylation. Values are expressed as means se; = 3. * 0.05 = 3. substrate (DPPC) concentration at varying p67phox concentrations (indicated in micrograms per milliliter by the number above each line). p67phox concentration. Intercept of the plot with the horizontal axis indicates p67phox concentration also was linear (Fig. 4= 3. = 6 for minus MJ33 and = 3 for plus MJ33. * 0.05 ?MJ33; ? 0.05 confirmation of the binding partners was demonstrated by ITC. These results demonstrated clearly that p67phox bound with much greater affinity to phosphorylated Prdx6, while phosphorylation of p67phox prevented an interaction. We next considered the physiological role of the interaction between p67phox and phosphoPrdx6. The presence of p67phox markedly inhibited the PLA2 activity of recombinant Prdx6 assayed em in vitro /em . Relevance of this interaction to intact cells was demonstrated using siRNA to knock down p67phox, thereby resulting in significant augmentation of the increase in PLA2 activity in response to Ang II. Inhibition by MJ33 provides evidence that the increased PLA2 activity with siRNA knockdown represents the PLA2 activity of Prdx6 (13, 20). We interpret this experiment as indicating that the increase in PLA2 activity following Ang II stimulation in control endothelial cells is restrained by the presence of p67phox. We postulate that inhibition of PLA2 activity by p67phox can terminate the signal for translocation of the cytoplasmic (NOX2) proteins and thereby limit the generation of potentially toxic metabolites of the PLA2 reaction. The p67phox that translocates to the plasma membrane is the phosphorylated protein, while the PLA2 turn-off signal Indacaterol requires nonphosphorylated p67phox. The present results suggest the following cycle related to Prdx6 and NOX2 activity. Stimulation of cells results in phosphorylation of Prdx6 that then binds to lipids in the cell membrane. The subsequent PLA2 activity results in liberation of products that Indacaterol signal for phosphorylation of cytosolic components (including p67phox) and Indacaterol activation of NOX2. Meanwhile, ECSCR p67phox (nonphosphorylated) binds to phosphoPrdx6, thereby terminating its PLA2 activity. A remaining question is how phosphoPrdx6 can be activated initially in the presence of cytosolic p67phox. We postulate.