Fig. 5

cPLA₂α activates NF-κB through activation of NOX2-NADPH oxidase in the BV-2 microglia cells under IFNγ stimulation. a A representative immunoblot analysis of the kinetics of cPLA₂α phosphorylation induced by 10 ng/ml IFNγ, out of three independent experiments. The intensity of phosphorylated cPLA₂α (p-cPLA₂α Ser-505) was quantitated by densitometry as described in Fig. 3a. b The BV-2 microglia cells were treated with 2 μm pyrrophenone (Pyrro) or 5 μm DPI for 1 h before stimulation with 10 ng/ml IFNγ for 4 h. Phosphorylated cPLA₂α (p-cPLA₂α Ser-505) intensity was quantitated by densitometry as described in Fig. 3a. The bar graphs are the mean ± SE from three independent experiments. c The effect of cPLA₂α inhibition on superoxide production in the unstimulated or stimulated BV-2 cells with 10 ng/ml IFNγ for 4 h detected by DHE reduction. Two micrometer pyropheonoe (Pyrro) or 200 μm apocynin (used as a positive control) were added to the cells 60 min before stimulation with IFNγ for 4 h. AS or sense (SE) were added 24 h prior to addition of IFNγ. DAPI staining shows cell nuclei. The intensity of reduced DHE was quantitated and expressed in the bar graph as arbitrary units. Scale bars large = 50 μm, insert = 20 μm. The bar graphs are the mean ± SE from three independent experiments. d Immunoprecipitation of p47^phox and phoshpo cPLA₂α (pcPLA₂α) in the membrane fraction of unstimulated microglia and stimulated with IFNγ for 4 h. Shown a representative immunoblot of three experiments. e Addition of 10 μM arachidonic acid togeher with IFNγ to the cells pre-treated for 24 h with antisense against cPLA₂α restored the expression of CD40 protein. Shown a representative immunofluorescence staining of CD40. DAPI staining shows cell nuclei. The intensity of CD40 was quantitated for the cell and expressed in the bar graph as arbitrary units. Scale bars = 50 μm. f FACS analysis of CD40 protein expression in the unstimulated or stimulated BV-2 cells with10 ng/ml IFNγ for 24 h in the absence or presence of 2 μm pyrrophenone (Pyrro) or 5 μm DPI added to the cells 1 h before stimulation. The bar graphs are the X-median ± SE from five independent experiments. g A representative immunoblot analysis of phosphorylated NF-κB p65 (p-NFκB p-p65(Ser-536) in unstimulated or stimulated BV-2 microglia by 10 ng/ml IFNγ for 4 h in the absence or presence of 2 μm pyrrophenone (Pyrro) or 5 μm DPI. The intensity of each p-NFκB p-p65(Ser-536) band after quantification as described in Fig. 3g. The bar graphs are the mean ± SE from three independent experiments. h A representative immunoblot analysis of phoshpo cPLA₂α and phospho NF-κB p-65 subunit and phosspho ERK1/2 in unstimulated or stimulated BV-2 microglia by 10 ng/ml IFNγ for 4 h in the absence or presence of 5 μM OU126. The bar graphs are the mean ± SE of the intensity of the quantitated phosphorylated forms divided by the non-phophorylated forms of three independent experiments. (***p < 0.0001)