Fig. 2.

SERT and NET TMD I Asp mutants do not alter transporter cell-surface expression. COS-7 cells were transfected with rSERT, D98E, D98G, or pcDNA3 vector (control), and cell-surface biotinylation experiments were performed as described in Materials and Methods. A, Immunoblots identifying rSERT and rSERT mutants using SERT-specific antibody CT-2B (Qian et al., 1995).B, Immunoblots identifying hNET and hNET D75E mutant using NET-specific antibody (Melikian et al., 1994). After blots were probed with the anti-SERT or anti-NET antibodies, membranes were stripped and reprobed with anti-calnexin antibody (StressGen). Because calnexin is a endoplasmic reticulum transmembrane protein, it is a marker for intracellular proteins. Low levels of calnexin bands (<0.5% of total) were detectable in the cell-surface lanes, suggesting only minor contamination of biotinylated (cell surface) fractions with intracellular proteins. Density of bands was determined using scanning densitometry (ImageQuant, Molecular Dynamics) with SERT total fractions normalized to total calnexin immunoreactivity. Data presented are representative of three separate experiments.