The cytosolic NADPH oxidase subunit p47phox and MAPK activation is mutually dependent on the ROS generation and cytokine production by s-Mtb-stimulated microglia. A) p47phox is required for s-Mtb-induced MAPK activation. BV-2 cells were transfected with wild-type p47phox (WT), dominant-negative p47phox (DN), or empty vector. The cells were then stimulated with 1% s-Mtb for 30 min, harvested, and subjected to Western blot analysis to detect total and phosphorylated ERK1/2 and p38. B) p47phox phosphorylation at Ser345 is required for s-Mtb-induced cytokine production. BV-2 cells were pre-treated with TAT-Ser345 peptide (20 or 40 μM) or TAT-scramble peptide (20 or 40 μM) and stimulated with 1% s-Mtb for 18 h. The supernatants were analyzed for TNF-α, IL-6, and IL-12p40 production by ELISA. Data are presented as the percentage of the control. Significant differences compared to cultures incubated with s-Mtb alone: **, P < 0.01; ***, P < 0.001. C) MAPK activation is essential for p47phox activation. After pretreatment for 30 min with inhibitors of either MEK1 (U0126; 5, 10, or 20 μM) or p38 (SB203580; 1, 5, or 10 μM), BV-2 cells were stimulated with 1% s-Mtb for 30 min. The cells were then harvested and subjected to Western blotting to detect phosphorylated pSer345 and p47phox. D) MAPK activity is required for s-Mtb-induced superoxide production. BV-2 cells were pretreated with U0126 (10 μM) or SB203580 (5 μM) for 30 min and then incubated with DHE (for superoxide detection) after stimulation with 1% s-Mtb for 30 min. The live cells were washed with serum-free medium and imaged using confocal microscopy. The images are representative of three independent experiments. M, medium only; D, 0.1% DMSO as a solvent control.