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Figure 5 | Journal of Neuroinflammation

Figure 5

From: Neuroimmune modulation following traumatic stress in rats: evidence for an immunoregulatory cascade mediated by c-Src, miRNA222 and PAK1

Figure 5

Modulation of PAK-1 signaling in cultured neurons. Rat cortical neurons were grown on coverslips for 10 days. Neurons were then immunostained using anti-c-Src and anti-PAK1 antibodies, and double-labeled cells were identified using a Leika Q500IW image analysis system (A). Neurons were treated with vehicle or PP2 for 30 min, and then assessed for microRNA222 (B) and PAK 1 (C) expression using real-time PCR and western blot, respectively. (D) Directed expression of c-Src in cultured neurons by transfection with adenovirus expressing active c-Src (CA c-Src). Endogenous c-Src activity was blocked using the synthetic inhibitor PP2, and association of PAK1 with IL-1RI was determined by immunoprecipitation assay. The immunoprecipitation antibody was anti-IL-1RI and the immunoblotting antibody was anti-PAK1. (E) The graph depicts expressions as percentages of controls. (F) Neurons were exposed to IL-1β and IL-1ra as described in Methods, and immunoprecipitation was used to analyze the association between PAK1 and IL-RI. The immunoprecipitation antibody was anti-IL-1RI and the immunoblotting antibody was anti-PAK1. The graph depicts expressions as percentages of controls (G). The results were normalized against an internal control and further normalized against the results obtained from control cultures. Data were analyzed by one-way ANOVA with Dunnett test as a post hoc test to assess comparisons.* P <0.05 vs Con, # P <0.05 vs c-Src, or IL-1β. Scale bars = 50 μm.

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