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Fig. 4 | Journal of Neuroinflammation

Fig. 4

From: NFκB signaling drives pro-granulocytic astroglial responses to neuromyelitis optica patient IgG

Fig. 4

The release of pro-granulocytic chemokines in response to stimulation with NMO IgG is NFκB-dependent. a CCL5, CCL2, CXCL1, and CXCL2 release in unstimulated cells (UNT) or following 6- or 24-h stimulation with 100 μg/mL NMO IgG (NMO) or CON IgG (CON) was measured by ELISA. The release of all four chemokines was significantly increased following stimulation with NMO IgG. Stimulation with control IgG did not result in release of any of the chemokines above baseline levels. Data shown are from 2 separate experiments performed in triplicate; each dot represents an individual well. The mean response (solid horizontal bar) and 95 % confidence intervals (error bars) are shown. Significance was determined by one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test (****p < 0.0001 versus CON IgG). b Fold inhibition of CCL5, CCL2, CXCL1, and CXCL2 release following stimulation with NMO IgG (100 μg/mL) for 6 h or 24 h after pretreatment with NFκB pathway inhibitors was calculated by comparison to cells stimulated with NMO IgG in the absence of inhibitor (amount released with NMO alone/release with NMO+ inhibitor); larger bars equal more robust inhibition. Inhibitor concentrations: 50 μM MG132, 30 μM BAY 11–7082, 30 μM SN50, 30 μM NEMO binding peptide (NBP). MG132 and BAY 11–7082 effectively inhibited CCL5, CCL2, and CXCL1 responses. All of the inhibitors exerted only limited inhibition of CXCL2 release, suggesting that this factor is induced by alternative signaling mechanisms. Data shown are from two separate experiments performed in triplicate. Error bars represent the 95 % confidence interval. p < 0.001 from three-way ANOVA comparing chemokines, time point, and inhibitors used for treatment

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