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Figure 6 | BMC Neuroscience

Figure 6

From: Expression, sorting and transport studies for the orphan carrier SLC10A4 in neuronal and non-neuronal cell lines and in Xenopus laevis oocytes

Figure 6

Localization and transport function of SLC10A4/NTCP chimeras in CAD cells. a The shown SLC10A4/NTCP chimeric constructs were used. All chimeras were generated based on the full length sequences of SLC10A4 (grey marked transmembrane domains and loops with continuous lines) and NTCP (white transmembrane domains and loops as dotted lines), both with c-terminal V5-tag. Potential glycosylation sites were marked by “Y”. b All constructs were transiently transfected in CAD cells and cellular localization was analyzed by immunofluorescence microscopy using rabbit anti-V5 antibody and donkey Cy3-labelled anti-rabbit secondary antibody. Nuclei were stained with DAPI. Whereas SLC10A4 showed a clear vesicle-like expression pattern, the immunofluorescence signals for NTCP, NtSLC10A4-NTCP, SLC10A4-CtNTCP, and NtNTCP-SLC10A4-CtNTCP were clearly directed to the plasma membrane. When the 75 N-terminal amino acids were deleted in SLC10A4, the 75ΔSLC10A4 protein retained its vesicle-like intracellular expression comparable with full-length SLC10A4. c The SLC10A4/NTCP chimeras were also used for transport studies after transient transfection into CAD cells with [3H]taurocholic acid and [3H]serotonin, each at 5 µM. These measurements were performed by incubating the cells for 60 min at 37°C in 250 µl cell medium with 50 µl sodium transport buffer containing the radiolabeled and non-radiolabeled compounds. NTCP and SERT were used as a positive control, and empty-vector transfected cells served as the negative control. After the uptake phase, cells were washed with ice-cold PBS, lysed, and subjected to scintillation counting. Data represent mean ± SD of representative experiments each with quadruplicate determinations. *Significantly different from control with p < 0.01.

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