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Figure 8


Fig. 8. PI3K activity is necessary for the axon elongation and branching induced by P2X7 inhibitors. (A) Hippocampal neurons (3 DIV) cultured in the presence or absence of BBG (5 µM) and/or the PI3K inhibitor LY-294002 (LY; 50 µM). LY and/or BBG were added after 1 DIV. Neurons were stained with an antibody against tyrosinated {alpha}-tubulin to define the neuronal morphology. Scale bar: 50 µm. (B) Quantification of axon length and the number of axon ramifications in neurons treated with BBG and/or LY. Graphs represent the mean ± s.e.m. from three independent experiments (n=150 neurons; ***P<0.001, ****P<0.0001, paired t-test). (C,D) Time course of Akt (C) and GSK3 (D) phosphorylation in hippocampal neuron extracts treated with BBG (5 µM) for 30 or 60 minutes. Graphs represent the mean ± s.d. from three different experiments. Note the significant increase in GKS3 phosphorylation after a 30-minute exposure (*P<0.05, paired t-test). Akt phosphorylation is also upregulated but this increase is not significant when compared with the controls. (E) Neurons treated with BBG display differences in the phosphorylation of the GSK3 substrate, tau. In the presence of BBG, the levels of the unphosphorylated tau-1 epitope augment whereas those of the hyper-phosphorylated epitope, PHF-1, diminish. The graph represents the tau-1:PHF-1 ratio in untreated (0 minutes) and treated (30 or 60 minutes) neurons (n=3, *P<0.001, paired t-test). (F) GSK3 phosphorylation in extracts of hippocampal neurons treated for 6 DIV with BBG (50 µM), Ip5I (1 µM) or PPADS (30 µM). Only exposure to BBG significantly increases GSK3 phosphorylation (*P<0.05, paired t-test). The graphs represent the mean ± s.d. from three independent experiments. (G) Neurons were treated after 1 DIV with ATP (1 mM) in the presence or absence of the GSK3 inhibitor AR-A014418 (20 µM). These neurons were then fixed at 3 DIV, and stained with anti-{alpha}-tubulin antibodies to analyze axon length and the number of axonal ramifications. The graphs represent the mean ± s.e.m. from three independent experiments (n=150; *P<0.05, ****P<0.0001, paired t-test). Scale bar: 50 µm.





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