Journal: BMC Neuroscience

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

doi: 10.1186/s12868-015-0174-2

Figure Lengend Snippet: Expression and subcellular localization of SLC10A4 in SH-SY5Y and CAD cells. a Relative SLC10A4 gene expression in SH-SY5Y cells after differentiation with TGF-ß1 + RA or BMP-2 + RA. Values represent mean ± SD of triplicate measurements. b Immunofluorescence analysis of the subcellular expression of the SLC10A4 protein in SH-SY5Y cells. Cells were either untreated (UD), or were differentiated with TGF-ß1 + RA or BMP-2 + RA over 4 days prior to immunolabeling. The SLC10A4 protein was detected with the anti-Slc10a4 1338 C antibody (1:1,000) and the Cy3-labelled anti-rabbit secondary antibody (1:800, red fluorescence) and nuclei were stained with DAPI (blue fluorescence). In all cases, the SLC10A4 protein showed a vesicle-like expression pattern within the perikarya and along the neurite-like cellular protrusions. c Even when a fluorescence-tagged SLC10A4-RFP construct was transiently transfected into SH-SY5Y cells, the SLC10A4-RFP protein showed a clear vesicular sorting pattern. d Relative Slc10a4 gene expression analysis in differentiated (Diff) and undifferentiated (UD) CAD cells. The values represent mean ± SD of triplicate measurements. e Endogenous expression of the SLC10A4 protein in CAD cells, cultivated in FCS containing medium (UD) or FCS-free medium (Diff). The SLC10A4 protein was detected with the anti-Slc10a4 1338 C antibody (1:500) and the Cy3-labelled secondary antibody (1:800, red fluorescence). For control, the primary anti-Slc10a4 antibody was omitted (control) or the antibody was pre-incubated with the immunizing peptide (peptide blocking). f Immunofluorescence detection of the SLC10A4 protein was performed with different SLC10A4-directed antibodies (green fluorescence): self-generated polyclonal rabbit anti-Slc10a4 1338 C antibody, rabbit anti-SLC10A4 Sigma Prestige antibody, rabbit anti-Slc10a4 Abnova antibody, and rabbit anti-SLC10A4 Abgent antibody. Membrane protein enriched fractions of the CAD cells were also subjected to Western Blot analysis with the same antibodies and revealed specific bands for the SLC10A4 protein at an apparent molecular weight of 30–32 kDa.

Article Snippet: PCR amplification was achieved with the TaqMan Gene Expression Assays Hs00293728 for human SLC10A4 and Mm00557788 for mouse Slc10a4 (Applied Biosystems).

Techniques: Expressing, Gene Expression, Immunofluorescence, Immunolabeling, Fluorescence, Staining, Construct, Transfection, Control, Incubation, Blocking Assay, Generated, Membrane, Western Blot, Molecular Weight

Journal: BMC Neuroscience

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

doi: 10.1186/s12868-015-0174-2

Figure Lengend Snippet: Transport measurements in transiently transfected HEK293 cells. HEK293 cells were transiently transfected with the indicated carriers SLC10A4, DAT, CHT1, or SERT, respectively. The uptake of 5 µM [ 3 H]dopamine, 5 µM [ 3 H]norepinephrine, 5 µM [ 3 H]choline, or 5 µM [ 3 H]serotonin was measured over the given time periods in the presence and absence of Na + (for CHT1 Na + was replaced by Li + ). Transport via DAT, CHT1, and SERT was blocked by the specific inhibitors nomifensine (10 µM), hemicholinium-3 (HC-3, 1 µM), and citalopram (2 µM), respectively. Values represent mean ± SD of representative experiments, each with quadruplicate determinations (n = 4). *Significantly different from control with p < 0.001. # Significantly different from positive uptake, p < 0.001.

Article Snippet: PCR amplification was achieved with the TaqMan Gene Expression Assays Hs00293728 for human SLC10A4 and Mm00557788 for mouse Slc10a4 (Applied Biosystems).

Techniques: Transfection, Control

Journal: BMC Neuroscience

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

doi: 10.1186/s12868-015-0174-2

Figure Lengend Snippet: Transport measurements in digitonin permeabilized SLC10A4-HEK293 and VMAT2-HEK293 cells. Prior to transport measurements, stably transfected human SLC10A4-HEK293 cells and human VMAT2-HEK293 cells were pre-incubated with 15 µM digitonin for 10 min for permeabilization. Then the uptake of 400 nM [ 3 H]serotonin, [ 3 H]norepinephrine, or [ 3 H]dopamine was measured over 10 min in the presence and absence of 5 mM ATP in the transport buffer. In addition to ATP, 2 µM of the potent VMAT2 inhibitor tetrabenazine (TBZ) or 5 µM of the proton ionophore carbonylcyanide-p-trifluoromethoxyphenylhydrazon (FCCP) were added to the transport buffer as indicated. After 10 min, the cells were washed with ice-cold PBS, lysed, and subjected to scintillation counting. The values represent mean ± SD of one representative experiment (for dopamine, n = 4) or two independent experiments (for serotonin and norepinephrine, n = 8). *Significantly different from control with p < 0.05. # Significantly different from positive uptake, p < 0.05.

Article Snippet: PCR amplification was achieved with the TaqMan Gene Expression Assays Hs00293728 for human SLC10A4 and Mm00557788 for mouse Slc10a4 (Applied Biosystems).

Techniques: Stable Transfection, Transfection, Incubation, Control

Journal: BMC Neuroscience

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

doi: 10.1186/s12868-015-0174-2

Figure Lengend Snippet: Transport measurements in Xenopus laevis oocytes. Xenopus laevis oocytes were injected with cRNA coding for human SLC10A4, SERT, DAT, or NTCP as well as mouse Oct1. Uptake of [ 3 H]serotonin, [ 3 H]histamine, [ 3 H]PREGS, [ 3 H]dopamine, [ 3 H]DHEAS, or [ 3 H]taurocholic acid, each at 1 µM, was measured over a time period of 10–60 min as indicated in the presence of sodium chloride in the transport buffer. SERT, Oct1, and DAT served as controls for the transport of [ 3 H]serotonin, [ 3 H]histamine, and [ 3 H]dopamine, respectively. NTCP was the reference carrier for PREGS, DHEAS and taurocholic acid. Afterwards, the oocytes were washed with ice-cold transport buffer, lysed and subjected to scintillation counting. The values represent mean ± SD of one representative experiment with n = 10 oocytes each. *Significantly different from control with p < 0.001.

Article Snippet: PCR amplification was achieved with the TaqMan Gene Expression Assays Hs00293728 for human SLC10A4 and Mm00557788 for mouse Slc10a4 (Applied Biosystems).

Techniques: Injection, Control

Journal: BMC Neuroscience

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

doi: 10.1186/s12868-015-0174-2

Figure Lengend Snippet: Transport measurements in stably transfected SLC10A4-HEK293 and NTCP-HEK293 cells after thrombin treatment. For the transport measurements, one part of the cells was pre-incubated with 1 U/200 µl thrombin over 3 h (+Thrombin), before the uptake of [ 3 H]DHEAS, [ 3 H]taurocholic acid, [ 3 H]PREGS, or [ 3 H]lithocholic acid (each at 300 nM) was measured over a time period of 10 min at 37°C. The cells were washed with ice-cold PBS, lysed, and subjected to scintillation counting. The values represent mean ± SD of two independent experiments each with triplicate determinations. *Significantly different from control with p < 0.001; n.s. not significantly different.

Article Snippet: PCR amplification was achieved with the TaqMan Gene Expression Assays Hs00293728 for human SLC10A4 and Mm00557788 for mouse Slc10a4 (Applied Biosystems).

Techniques: Stable Transfection, Transfection, Incubation, Control

Journal: BMC Neuroscience

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

doi: 10.1186/s12868-015-0174-2

Figure Lengend Snippet: 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 [ 3 H]taurocholic acid and [ 3 H]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.

Article Snippet: PCR amplification was achieved with the TaqMan Gene Expression Assays Hs00293728 for human SLC10A4 and Mm00557788 for mouse Slc10a4 (Applied Biosystems).

Techniques: Construct, Generated, Glycoproteomics, Transfection, Immunofluorescence, Microscopy, Staining, Expressing, Clinical Proteomics, Membrane, Positive Control, Plasmid Preparation, Negative Control, Control

Journal: BMC Neuroscience

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

doi: 10.1186/s12868-015-0174-2

Figure Lengend Snippet: Transport studies in HEK293 cells

Article Snippet: PCR amplification was achieved with the TaqMan Gene Expression Assays Hs00293728 for human SLC10A4 and Mm00557788 for mouse Slc10a4 (Applied Biosystems).

Techniques:

Journal: BMC Neuroscience

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

doi: 10.1186/s12868-015-0174-2

Figure Lengend Snippet: Primers used for full-length carrier cloning

Article Snippet: PCR amplification was achieved with the TaqMan Gene Expression Assays Hs00293728 for human SLC10A4 and Mm00557788 for mouse Slc10a4 (Applied Biosystems).

Techniques: