Fig. 6

Endogenously produced TNF-α regulates cPLA₂α activation in the BV2 cells under IFNγ stimulation. a A representative immunoblot analysis of the kinetics of cPLA₂α activation detected by its phosphorylated form induced by 10 ng/ml TNF-α in BV-2 microglia lysates. A representative immunoblot analysis of a dose-dependent activation of the cPLA₂α (b) and of NF-kB (c) detected by their phosphorylated induced by TNF-α in BV-2 microglia lysates. Representative immunoblot analysis of (d) cPLA₂α phosphorylation on Ser-505 and (e) NF-kB p-65 phosphorylation on 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 50 ng/ml TNF-α-neutralizing antibody (anti-TNF-α) added to the cells 60 min before stimulation with IFNγ stimulation. The intensity of each p-cPLA₂α(Ser-505) or p-NFκB p65(Ser-536) band after quantification by densitometry was divided by the intensity of each cPLA₂α or NFκB p65 band, respectively, and expressed as arbitrary units. The results are the mean ± SE from four experiments. f Immunofluorescence analysis of CD40 protein expression in unstimulated or stimulated BV-2 microglia with 10 ng/ml IFNγ for 24 h in the absence or presence of 50 ng/ml TNF-α-neutralizing antibody or stimulated by 10 ng/ml TNF-α. Scale bars = 50 μm. The intensity of CD40 was quantitated and expressed in the bar graph as arbitrary units. The bar graph is the mean ± SE from four independent experiments. DAPI staining shows cell nuclei. (***p < 0.0001, n.s. not significant)