Neurturin Evokes MAPK-Dependent Upregulation of Egr4 and KCC2 in Developing Neurons
Regulation of KCC2 expression by Neurturin. (a) Representative Western blot analysis of Neurturin-induced ERK1/2 phosphorylation. Lysates of div5-dissociated cultures were collected 5–20 min after Neurturin (50 ng/mL) application. In some cases cultures were pretreated with MEK inhibitor U0126 (20 μM) 30 min before Neurturin application. (b) Egr4 mRNA level in div5–div10 dissociated cultures 1-2 hours after Neurturin (50 ng/mL) application as detected by real time PCR (). Nontreated control value was set to 1. (*, **, ***). Error bars represent SEM. (c) Representative Western blot analysis and quantification of KCC2 expression in organotypic hippocampal slice cultures treated with NTRN (n = 4–8). Organotypic cultures were treated with 10 ng/mL and 50 ng/mL Neurturin. Data are normalized to the value in nontreated controls (*, **, ***). Error bars represent SEM. (d) Representative Western blot analysis and quantification of KCC2 expression in dissociated hippocampal cultures treated with Neurturin (50 ng/mL) (n = 3–7). Dissociated cultures were treated with Neurturin at div1, div8 and div15 and analyzed 3 days after the treatment. Data are normalized to the value of non-treated controls of the corresponding age (*, **, ***). Error bars represent SEM. (e) Representative semiquantitative RT PCR from cDNA of different age cultures for RET, GFRα2, and GAPDH (used as internal standard) and summarized results of 3 similar PCRs. The data show that dissociated hippocampal neurons express detectable levels of growth factors receptors at all ages tested. Data are normalized to div4 value. Error bars represent SEM.
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