Figure 6

Overexpression of Dock3 inhibits glutamate-induced intracellular Ca²⁺elevation and prevents glutamate-induced apoptosis in RGCs. (A) RGCs from WT or Dock3-transgenic (Tg) mice were labeled with Fluo-8 acetoxymethyl ester for 30 min, and then 300 μM glutamate (Glu) was added. Fluorescence-ratio images are displayed in pseudocolor, as indicated by the color bar on the right side of the images. Pseudocoloring represents ratios between 0 and 2, corresponding to 1, which is defined as the basal fluorescence intensities before Glu stimulation. Left and right panels show ratio images for WT and Dock3-Tg, respectively. Scale bar: 100 μm. (B) Changes in Fluo-8 fluorescence are expressed as ΔF/F0, where F0 is the basal fluorescence intensity before Glu stimulation. Glu was added as indicated. Data represent the mean ± SE from eight independent experiments. (C) Fragmented or shrunken nuclei were detected by Hoechst staining 24 hr after control (HBSS) or glutamate (Glu) treatment. **p < 0.0001 for WT Glu vs. Dock3-Tg Glu. WT, wild-type mice; Dock3-Tg, Dock3 transgenic mice. (D) N2A cells were transfected with NR1 and NR2D with (Dock3+) or without Dock3 (Dock3-). Surface proteins were biotinylated using sulfo-NHS-SS-biotin, immunoprecipitated with streptavidin beads and probed with NR2D antibodies. Data are from a single experiment, which is representative of five experiments that yielded similar results. (E) Relative ratio of biotinylated protein to total protein. *p < 0.05. Data represent the mean ± SE from five independent experiments.