Effect of insCGTT type on distribution of NR3B in HEK293T cells.

<p><b>A</b>, HEK293T cells transfected with GluR1 tagged with GFP at the extracellular N-terminus or intracellular C-terminus were stained with a GFP antibody under non-permeabilized conditions on ice, as verification of the specificity of cell surface receptor detection using immunostaining. <b>B</b>, HEK293T cells transfected with GFP-tagged NR3B major type or insCGTT type (green) were stained with GFP antibody under non-permeabilized condition to detect the cell surface population (surface GFP, red). <b>C</b>, Quantification of the surface levels of NR3B major type and insCGTT type with and without NR1. Because expression level of major type and insCGTT were different, the level was normalized by total GFP fluorescence in each cell. Data are expressed as a normalized value to that of major type alone. n = 55, 52, 59, and 52 for GFP-NR3B major type only, GFP-NR3B major type with NR1, GFP-NR3B insCGTT type only, and GFP-NR3B insCGTT type with NR1, respectively. *: p < 0.05; **: p < 0.01; ns: not significant. <b>D</b>, Surface biotinylation of NR3B major type and insCGTT type. GFP-tagged NR3B major type or insCGTT type was coexpressed with NR1 in HEK293T cells and the surface population was labeled by biotin. NR3B types in both total (T) and biotinylated surface population (S) were detected by anti-GFP antibody. Total fractions represent 1/8 of the starting material applied in surface fractions. α-Tubulin (Tub), an intracellular protein, was not biotinylated, confirming specificity of surface biotinylation.