Fig. 8. Dok-4 induces neurite outgrowth in rat hippocampal neurons. (A) Expression of rat Dok-4 and RET in rat hippocampal neurons. Total RNA was isolated from PC12 cells and rat hippocampal neurons, and RT-PCR performed using rat Dok-4-and RET-specific primers as described in Materials and Methods. ß-actin was amplified as a control. (B) Hippocampal neurons were transfected with GFP, GFP-Dok-4 wild-type, GFP-Dok-4 Y220F mutant, GFP-RapGAP (SPA-1) or GFP-RapN17, incubated for 72 hours, and visualized by confocal microscopy. Boxes denote dendrites of neurons identified by MAP2 expression (lower panels). (C) Hippocampal neurons transfected with the indicated constructs were incubated for 72 hours with or without GDNF (50 ng/ml), and dendrite length quantified. Mean of the longest dendrite was determined for each culture from measurements of 50 hippocampal neurons for three different experiments. Each data point represents the mean ± s.e.m.