Figure 1.

Confocal microscopy and [³H]taurocholate (TC) efflux studies of human hOSTα and truncated β co-expressed in stably transfected MDCK cells. (A) Confocal fluorescence images: The cells were grown on glass coverslips and fixed with cold 100% methanol. In stably transfected MDCK cells, the hOSTα were co-expressed with a GFP fused wild type (β-GFP), C-terminal truncated (β-CT-GFP) or N-terminal truncated (β-NT-GFP) hOSTβ. TC transport activity of cells transfected with human organic solute transporter (hOST): Na⁺-independent [³H]TC efflux (B) and Na⁺-dependent [³H]TC uptake (C) were measured in stably transfected MDCK cells that were transfected with rat Asbt, human OSTα and wild type (β), or C-terminal truncated (β-CT) or N-terminal truncated (β-NT) hOSTβ cDNA. Data represent mean ±S.E. (bars) of triplicate determinations from at least three different cell culture preparations. Asterisks (*, p<0.05) indicate significant differences from wild type hOSTβ co-transfected cells. Messenger RNA levels of rAsbt, wild type and truncated hOSTα and β constructs in stably transfected MDCK cells were obtained using the reverse transcription-polymerase chain reaction (RT-PCR) (D) (see methods), and the reaction products were visualized by staining with ethidium bromide. Figure (C) and (D) indicate that these stably transfected MDCK cells have similar gene expression level for rAsbt, wild type and truncated hOSTα and β, and equally TC uptake activity.