Figure 4.

Ethanol increases surface density of DAT. Ethanol-induced changes in the cell surface density of wild-type DAT, containing an N-terminal eGFP tag, in DAT HEK cells was determined using cell surface biotinylation assays. Cells were incubated in KRH buffer in the absence or presence of 100 mM ethanol for 1h at 37°C. (a) Representative immunoblots of DAT and calnexin in biotinylated DAT HEK cell lysate from four separate experiments. Shown are the total extracts (total DAT 55−125 kDa) and biotinylated extracts (cell surface DAT 90−125 kDa) from DAT HEK cells with indicated treatments. Blots were stripped and reprobed with the antibody for endogenous intracellular control protein, calnexin. (b) Mean optical density (O.D.) of total DAT in ethanol-treated (empty bars) DAT HEK cells did not significantly change compared to total DAT in untreated cells (filled bars), while the cell surface DAT O.D. was significantly increased in ethanol treated cells. **p <0.01 compared to untreated surface DAT O.D. (Student's t test). (inset) Quantification of the percent change in cell surface DAT populations between ethanol treated and untreated cells. Ethanol significantly increases surface DAT populations 40−50% (expressed as a percentage relative to the total amount of DAT in the respective lysate (untreated or ethanol-treated) and normalized to untreated surface DAT). Data are expressed as normalized values ± SEM of four experiments. *p < 0.05 compared with untreated surface DAT population (Student's t test).