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. 2014 Jun 3;289(30):20773–20787. doi: 10.1074/jbc.M114.550632

FIGURE 2.

FIGURE 2.

Rab17 is required for the surface expression of GluK2 without affecting its traffic to the dendrites. A, representative images of the distribution of surface-expressed Myc-GluK2 in developing hippocampal neurons. At 8 DIV, rat hippocampal neurons were co-transfected with pCDNA3-Myc-GluK2 and pEGFP-C2. At 14 DIV, the neurons were fixed and subjected to immunocytochemistry with antibodies against Myc (surface; black) and MAP2 (red) as a dendrite marker. The arrows and arrowheads point to axons and dendrites, respectively. Panels a–c are magnified views of the boxed areas in the top right panels. Bar, 10 μm. B, quantification of the ratio of surface-expressed Myc-GluK2 between soma, dendrite, and axon (n = 10) as shown in A. C, representative images of surface expression level of Myc-GluK2 in the Rab17-shRNA-transfected neurons. At 8 DIV, rat hippocampal neurons were transfected with pCDNA3-Myc-GluK2 together with pSilencer-CMV-EGFP-Control, pSilencer-CMV-EGFP-shRab17, or pSilencer-CMV-EGFP-shRab17 and pCMV-Rab17SR. At 14 DIV, neurons were fixed and subjected to immunocytochemistry with antibodies against Myc (surface, red and total, green). GFP image was replaced black in this figure. Bar, 10 μm. D, quantification of the surface expression level of Myc-GluK2 of control-shRNA-transfected neurons (n = 10) and Rab17-shRNA-transfected neurons (n = 10) as shown in C. The rate of surface expression level of Myc-GluK2 was calculated by dividing the surface Myc-GluK2 fluorescence intensity by the total Myc-GluK2 fluorescence intensity. A.U., arbitrary units. **, p < 0.0025. E, magnified views of the boxed areas in panels a–d of C. Bar, 10 μm. F, quantification of the total Myc-GluK2 fluorescence intensity in the dendrites of control-shRNA-transfected neurons (n = 10) and Rab17-shRNA-transfected neurons (n = 10) as shown in E. Total level of Myc-GluK2 in the dendrites was calculated by dividing the Myc-GluK2 fluorescence intensity by the dendrite length. A.U., arbitrary units. N.S., not significant. G, representative images of Myc-GluK2 and EGFP-Rab17 in neurons. At 8 DIV, rat hippocampal neurons were co-transfected with pEGFP-Rab17 and pCDNA3-Myc-GluK2. At 14 DIV, the neurons were fixed and subjected to immunocytochemistry with antibodies against Myc (red). The bottom panels are magnified views of the boxed areas in the upper panels. Bar, 5 μm.