Expression of Bsep-GFP in MDCK cells and HEK 293 cells. (a) After transient transfection with Bsep-GFP, MDCK cells were processed for immunofluorescence using antibodies against rat Bsep, gp-135, and NaK-ATPase, and imaged to locate the GFP fusion protein and apical and basolateral membranes of MDCK cells. In each panel, the top part shows the en face image and the bottom part shows the Z-sectioning image. Bsep-GFP colocalized with gp-135 but was distinct from NaK-ATPase. Bar, 5 μm. (b) Bsep-GFP was expressed in either a stable MDCK cell line or HEK 293 cells by transient expression. Both en face and Z-sectioning images of the GFP fusion protein are presented. Bar, 5 μm. Total membrane and cytosol fractions were prepared from (c) MDCK cell lines either stably expressing Bsep-GFP or expressing GFP alone, or (d) from transfected and mock-treated HEK 293 cells. These fractions (100 μg protein per lane) and a rat liver plasma membrane preparation (LPM) (100 μg protein per lane) were separated on a 6.5% Laemmli gel, and Bsep-GFP was detected by Western blotting using an antibody against rat Bsep. The position of Bsep-GFP in the total membrane fraction of MDCK cells is indicated by the arrow. Rat Bsep and Bsep-GFP have apparent molecular weights of approximately 160 kDa and 190 kDa, respectively.