Fig. 3

Plg receptors are required for induction of cytokine expression in N-astrocytes by Plg. a Specific binding of Plg-594 to N-astrocytes. The presented graph shows total binding, binding in the presence of εACA (non-specific), and specific binding, which is defined as binding that is displaced by εACA (mean of three separate binding studies). b–d N-astrocytes were treated with Plg (0.2 μM), Plg plus tPA (12 nM) or vehicle, in the presence or absence of 10 mM εACA for 6 h. RT-qPCR was performed to determine TNFα, IL-1β and CCL2 mRNA (mean ± SEM; n = 3; ***p < 0.001, **p < 0.01; one-way ANOVA with Tukey’s post hoc test. Statistical analysis is relative to the vehicle control). e–g N-astrocytes were treated with Plg (0.2 μM), Plg plus tPA (12 nM) or vehicle, in the presence of 10 μg/mL α-enolase-specific antibody, antibody that targets the Plg-binding site in actin (25 nM), or non-specific IgG for 6 h. RT-qPCR was performed to determine TNFα, IL-1β, and CCL2 mRNA. The ability of each antibody to inhibit cytokine expression in response to the eliciting agent (% inhibition) is shown (mean ± SEM; n = 4; **p < 0.01, *p < 0.05; two-way ANOVA with Tukey’s post hoc test). (h) N-astrocytes were treated with Plg (0.2 μM), Plg plus tPA (12 nM), or vehicle in presence and absence of α-enolase-specific antibody (10 μg/mL) for 1 h. Immunoblot analysis was performed to detect phospho-IκBα, IκBα, and β-actin