Figure 3.

GATA1 over-expression in cortical neurons decreases Rab4b expression and reduces dendrite branching. (a) rAAV-GATA1-EGFP vector construction. ITR, inverted terminal repeats; CMV, cytomegalovirus promoter; hGH-polyA, human growth hormone polyA signal (b) Levels of Gata1 protein, determined by western blot, are increased in response to rAAV-GATA1 infection; note that the size is shifted due to expression of conjugated GATA1-EGFP. (c) rAAV-GATA1 expression decreases levels of Rab4bdetermined by qRT-PCR. Cells were transfected at DIV5 and extracted at DIV19. Results of qPCR analysis of Rab4b represent the mean ± S.E.M. (n = 4). *P <0.05 compared to vector alone (unpaired t-test). (d, e) Images of cortical neurons infected with control virus (left panels) or rAAV-GATA1-EGFP (right panels) and stained with antibodies to GFP (d, scale bar = 20 µm; e, scale bar = 10 µm). (f) Cells were infected with control virus (left panel) or rAAV-GATA1-EGFP (right panel) as described above but were stained for the dendritic marker MAP2 (scale bar = 20 µm), which was also examined by (g) western blot analysis (n = 3). *P <0.05 compared to vector alone (unpaired t-test). (h) Representative picture of Sholl analysis and quantitative results of the number of neuronal process intersections (scale bar = 20 µm). Results represent the mean ± S.E.M. (n = 7 for control vector, n = 4 for GATA1). *P <0.01 compared to vector alone (unpaired t-test).