Expression and transport activities of Tyr117mutants. A, COS-7 cells were transiently transfected with expression vectors for T7-tagged wild-type or mutant mSlc10a2. Whole cell lysates were examined by western blotting using anti-T7 antibody (upper). Whole cell lysate of untransfected cells was loaded as a control (ctrl). Filled and open arrows indicate core-glycosylated and fully glycosylated forms of T7-mSlc10a2, respectively. T7-tag constructs yield lower levels of the fully glycosylated form compared to EGFP-fusion constructs. Circles indicate apparently nonspecific bands. The membrane was stripped and reprobed with anti-β-actin antibody (lower). B, Cell surface expression was measured by surface enzyme-linked immunosorbent assay. Data are expressed as mean and SE of a sextuplicate experiment. C, COS-7 cells transiently expressing wild-type or mutant mSlc10a2 were incubated with 0.02 μM 3H-TCA (370 GBq/mmol; American Radiolabeled Chemicals) for 5 min in the presence of 100 mM NaCl or 100 mM choline chloride, and Na+-dependent uptake was calculated by subtracting the uptake in the presence of 100 mM choline chloride from that in the presence of 100 mM NaCl. Na+-dependent uptake data are expressed as mean and SE of 3 independent experiments. #, Na+-dependent uptake is significantly different from that of wild-type Slc10a2 (p < 0.05). D, Kinetic analysis of Na+-dependent TCA transport by wild-type and Y117S. Data are expressed as the mean and SE of 3 independent experiments. Circles, wild type; squares, Y117S. E, Na+-dependent TCA uptake by the wild-type mSlc10a2 and Tyr117 mutants are plotted against the van der Waals volume of the residue at position 117.