FIGURE 2.

Expression and localization of Rab17 protein in the hippocampal neuron are developmentally regulated. A, specificity of the anti-Rab17 antibody used in this study. COS-7 cells were transfected with a vector encoding FLAG-tagged Rab5A, Rab17, or Rab20, and 2 days after transfection, the cells were lysed and subjected to immunoblot analysis with anti-Rab17 antibody (upper panel) and anti-FLAG tag antibody (lower panel). Note that Rab17 antibody specifically recognized Rab17 (upper panel, lane 2) but did not recognize its related isoforms, Rab5A and Rab20 (upper panel, lanes 1 and 3). B and C, Rab17 expression is developmentally regulated (B) in the mouse hippocampus and (C) in primary mouse hippocampal neuron cultures. Tissue homogenates (50 μg) of mouse hippocampus from embryonic day 18 (E18) to adult mice (B) or lysates of murine hippocampal neurons at 7, 11, 14, 21, or 28 DIV (C) were analyzed by immunoblotting with anti-Rab17 antibody (upper panels) and anti-actin antibody (lower panels). The asterisks indicate nonspecific bands. The positions of the molecular mass markers (in kilodaltons) are shown on the left. D and E, at 2 DIV mouse hippocampal neurons were transfected with a vector encoding EGFP, and the neurons were fixed at 3 DIV (D) or 11 DIV (E) and subjected to immunocytochemistry with antibodies against GFP (green), Rab17 (red), and MAP2 (blue). The bottom three panels (panels a–c) are magnified views of the boxed areas in the upper left panels. Note that at 3 DIV, endogenous Rab17 signals were specifically observed in the cell body, and they were hardly observed in the dendrites and axons, whereas at 11 DIV some Rab17 signals were clearly observed in the dendrites, but they were rarely observed in the axons. Bars, 50 μm (top panels in D and E) and 10 μm (middle and bottom panels in D and E).