Surface expression of hDAT and mutant hDAT in HEK 293T cells. A, cell
surface expression of hDAT and mutant hDAT proteins was measured by
biotinylation of cell surface proteins with sulfo-NHS-SS-biotin as described
under Materials and Methods. Biotinylated proteins were isolated
using avidin beads and analyzed by Western blotting using a monoclonal
anti-DAT antibody. The position of the 100-, 75-, and 50-kDa molecular mass
markers is shown. B, quantitation of the biotinylated surface and total (in
lysate) hDAT proteins in hDAT and mutant hDAT cells. The expression of the
constructs was measured by Scion Image software. Mean arbitrary density units
were plotted (n = 6) for the surface hDATs (biotinylated) and total
hDAT (lysate) for the hDAT, T62A-, and T62D-hDAT constructs. *,
one-way ANOVA, p < 0.002; in post-hoc Tukey-Kramer analysis,
p < 0.01 for hDAT versus T62D-hDAT, p < 0.05 for
T62A-hDAT versus T62D-hDAT. For lysate totals, one-way ANOVA, p <
0.001; in post-hoc Tukey-Kramer analysis, p < 0.001 for hDAT
versus T62D-hDAT; p < 0.05 for T62A-hDAT versus T62D-hDAT.