FIGURE 8.

Difopein inhibition of 14-3-3 binding to GluN2C reduces GluN2C surface expression. A, hippocampal neurons were co-transfected with 3x FLAG-tagged GluN2C, eCFP empty vector, or eCFP-difopein as indicated. Two days after transfection, cells were incubated with monoclonal anti-FLAG antibody for 30 min at room temperature. Cells were fixed and incubated with Alexa 647-conjugated anti-mouse secondary antibody (depicted in red for optimal visual contrast effect) to visualize the surface receptors. Cells were then washed, permeabilized, and labeled with polyclonal anti-FLAG antibody and Alexa 488-conjugated anti-rabbit secondary antibody (green) to visualize intracellular pool of receptors and with anti-GFP antibody and Alexa 405-conjugated anti-rabbit secondary antibody (gray) to visualize the difopein-transfected cells. The bottom panels display representative zoomed in higher magnification images of 10 μm dendritic segments. Scale bars = 10 μm. B, data were quantified by measuring ratios of surface GluN2C/intracellular GluN2C using ImageJ software. Data represent means ± S.E. (n = 9 cells per group; n = 27 dendritic segments per group; **, p < 0.001).