FIGURE 4.

14-3-3 isoforms differentially regulate GluN2C forward trafficking in HeLa cells. A, HeLa cells were co-transfected with GFP-GluN2C (containing a GFP protein tag in the extracellular N-terminal domain), GluN1–1a-IRES-DsRed (channel turned off) and Myc empty vector, Myc-14-3-3ϵ WT, Myc-14-3-3ζ WT, Myc-14-3-3ζ S145A/Y178H/C189I, Myc-14-3-3σ WT, or Myc-14-3-3σ A147S/I191C. Two days after transfection, cells were incubated with anti-GFP antibody for 30 min on ice. Cells were fixed and incubated with Alexa 647-conjugated anti-rabbit secondary antibody (depicted in red for optimal visual contrast effect) to visualize the surface receptors. Cells were then washed, permeabilized, and labeled with anti-GFP antibody and Alexa 488-conjugated anti-rabbit secondary antibody (green) to visualize intracellular pool of receptors and anti-Myc monoclonal antibody and Alexa 405-conjugated anti-mouse secondary antibody (gray) to visualize the 14-3-3-transfected cells. B, data were quantified by measuring ratios of surface GluN2C/intracellular GluN2C receptors using ImageJ software. Data represent means ± S.E. (n = 12 cells/group; **, p < 0.001; *, p < 0.05).