FIGURE 11.

Interaction of Oatp1d1-WT and Oatp1d1-mCitrine. A, cell localization of Oatp1d1 tagged on the N terminus with mCitrine (1d1/mCitrine) and cotransfection of Oatp1d1 without mCitrine and with mCitrine tag in a 3:1 ratio (1d1wt+1d1/mCitrine) obtained with immunofluorescence analysis by confocal microscopy. Plasma membranes are stained in red after binding of primary antibody Na,K-ATPase and Cy3-conjugated IgG secondary antibody (all anti-mouse). Nuclei are stained in blue with DAPI. White arrows indicate cell membrane localization of Oatp1d1. If the protein is localized to the membrane, the color turns to orange because of the overlap of green signal from the mCitrine tag and red stained plasma membranes. B, uptake of LY into the Oatp1d1-overexpressing cells in comparison with the HEK293 transiently transfected with either the empty vector pCS2-mCitrine or the Oatp1d1-mCitrine construct and after the cotransfection of Oatp1d1 without mCitrine and with mCitrine tag in a 3:1 ratio (Oatp1d1/Oatp1d1-mCitrine, 3:1). Transiently transfected HEK293 cells were incubated for 15 min with LY at 37°C. The reaction was stopped with two washing cycles in ice-cold PBS, and the cells were lysed in 0.1% SDS for 30 min. Data are expressed as -fold level of the LY uptake relative to the control (HEK293 cells transfected with the pcDNA3.1 empty vector). Each value represents the mean ± S.E. (error bars) from triplicate determinations.