Figure 3

Transient expression of wild-type and uncharged polar residue mutants of mSlc10a2 and cell surface biotinylation. A, Twenty micrograms of whole cell lysates of COS-7 cells transiently expressing wild-type or mutant mSlc10a2 fused with EGFP were subjected to 7.5% SDS-PAGE and western blotting was performed using anti-GFP antibody. Consistency of protein loading was confirmed by Ponceau S staining of the blotted PVDF membrane (not shown). B, COS-7 cells transiently expressing wild-type or mutant mSlc10a2 were incubated with 0.1 μM³H-TCA (185 GBq/mmol; Perkin-Elmer) for 5 min in the presence of 100 mM NaCl (shaded columns) or 100 mM choline chloride (open columns). Each column and error bar represents the mean and SE of 3 independent experiments, respectively. *, Uptake in the presence of NaCl is significantly higher than that in the presence of choline chloride (p < 0.05); #, Na⁺-dependent uptake is significantly different from that of wild-type Slc10a2 (p < 0.05). C, Na⁺-dependent uptake of TCA was calculated by subtracting the uptake in the presence of 100 mM choline chloride from that in the presence of 100 mM NaCl. Data represent the mean and SE of 3 independent experiments. D, Left panel, Whole cell lysates and biotinylated fractions of COS-7 cells transiently expressing wild-type or mutant mSlc10a2 were subjected to western blot analysis. The image is representative of 3 independent experiments. The graph shows the densitometry of bands ranging from 65–90 kDa expressed as the mean and SE of 3 independent experiments. Right panel, Western blot analysis of whole cell lysates and biotinylated fractions of untransfected (ut) and empty vector-transfected (v) COS-7 cells. Because the migration rate of EGFP (26.9 kDa) encoded by empty vector is smaller than that of EGFP-fused mSlc10a2, proteins were resolved on a higher concentration (12.5%) of SDS-polyacrylamide gel. E, Na⁺-dependent TCA uptake by wild-type and mutant mSlc10a2 were normalized to their cell surface expression.