FIGURE 5.

Co-expression of 14-3-3 isoforms does not affect GluN2C endocytosis in HeLa cells. A, an endocytosis assay was carried out using HeLa cells co-transfected with GFP-GluN2C, GluN1–1a-IRES-DsRed, and Myc-14-3-3ζ WT, Myc-14-3-3ζ S145A/Y178H/C189I, Myc-14-3-3σ WT, Myc-14-3-3σ A147S/I191C, or empty Myc vector. To visualize surface receptors, cells were incubated with anti-GFP antibody on ice, washed, and returned to conditioned medium for 30 min at 37 °C to allow receptor internalization. Cells were fixed and surface-expressed proteins were labeled with Alexa 647-conjugated secondary antibody (depicted in red). After permeabilization, internalized receptors were labeled with Alexa 488-conjugated secondary antibody (green) and Myc-14-3-3 was labeled with anti-Myc antibody and Alexa 405-conjugated anti-mouse secondary antibody (depicted in gray). B, data were quantified by measuring ratios of internalized/surface GluN2C receptors using ImageJ software. Data represent means ± S.E. (n = 15 cells/group; n.s. denotes no significant difference between groups).