Journal: Frontiers in Neuroscience

Article Title: Stretch stress propels glutamine dependency and glycolysis in optic nerve head astrocytes

doi: 10.3389/fnins.2022.957034

Figure Lengend Snippet: Optic nerve head astrocyte characterization . (A) Isolated optic nerve head (ONH) astrocytes were immunolabeled using antibodies against GFAP (green), β-tubulin (red, upper panels), and Iba1 (red, lower panels) to determine the purity of the cell cultures. DAPI was used to stain cell nuclei (blue). Neither β-tubulin nor Iba1 was detected in the cultures. Scale bar = 10 μm. (B) Sections of retina with ONH and optic nerve attached served as positive controls for the immunolabeling in (A) . Both β-tubulin and Iba1 were observed in the mouse ONH and optic nerve. Scale bar = 25 μm. (C) Quantification of β-tubulin and Iba1 protein in the optic nerve head (ONH) and isolated ONH astrocytes using capillary electrophoresis. Neither β-tubulin nor Iba1 was detected in the ONH astrocyte cultures (square symbols); n = 3 biological replicates per group. (D) Quantitative PCR for a number of glial transcripts and transcripts for retinal ganglion cells ( Rbpms ), microglia ( Iba1 ), and oligodendrocytes ( Mbp ) was used to further characterize the ONH astrocytes. Rbpms , Iba1 , and Mbp were not detected in mRNA isolated from control or stretched ONH astrocytes (ND). There were no differences in transcripts for Gfap , Slc1a3 (glutamate-aspartate transporter), Glud1 (glutamate dehydrogenase-1), and Glul (glutamine synthetase). There were significant differences in the Gja1 transcript levels, with stretched ONH astrocytes showing greater fold change as compared to control (* p = 0.0152, n = 3). Gja1 encodes Connexin-43; n = 3 biological replicates per group. (E) GFAP protein, increased when astrocytes undergo hypertrophic reactivity, did not statistically differ between control and stretched ONH astrocytes; n = 3 biological replicates per group. (F) S100β protein, also upregulated in reactive astrocytes, did not statistically differ between control and stretched ONH astrocytes; n = 3 biological replicates per group.

Article Snippet: Fully validated TaqMan assays used in this analysis: Actb (Mm02619580_g1), Gfap (Mm01253033_m1), Glud1 (Mm00492353_m1), Glul (Mm00725701_s1), Slc1a3 (Mm00600697_m1), Rbpms (Mm00803908_m1), Mbp (Mm01262037_m1), Iba1 (Mm00479862_g1), Gja1 (Mm00439105_m1).

Techniques: Isolation, Immunolabeling, Staining, Electrophoresis, Real-time Polymerase Chain Reaction

Journal: Frontiers in Neuroscience

Article Title: Stretch stress propels glutamine dependency and glycolysis in optic nerve head astrocytes

doi: 10.3389/fnins.2022.957034

Figure Lengend Snippet: Protein changes in ONHAs corroborate bioenergetics data . (A) Glucose transporter-1 protein levels in Stretched ONH astrocytes are significantly higher than Control (* p = 0.0225, n = 7 Control, n = 8 Stretch). Retinal lysate from a 2 month-old mouse was used as a positive control for each protein analyzed, while negative control was the signal obtained when no primary antibody was included in the capillary. (B) Lactate dehydrogenase-A, the astrocyte-specific isoform of the enzyme that catalyzes the interconversion of pyruvate and lactate, has equivalent protein levels in Control and Stretch ONH astrocytes. (C) Glucose-6-phosphate dehydrogenase, the enzyme that shunts glucose into the pentose phosphate pathway, is no different in Control and Stretch ONH astrocytes. (D) Glutamine synthetase, the enzyme that synthesizes glutamine from glutamate, is no different in Control and Stretch ONH astrocytes. (E) The monomeric form of glutamate-aspartate transporter (GLAST) has significantly higher protein levels in the Stretch as compared to the Control ONH astrocytes ( p = 0.020; n = 4 Control, n = 5 Stretch). (F) GLAST dimer protein levels are no different in Control and Stretch ONH astrocytes.

Article Snippet: Fully validated TaqMan assays used in this analysis: Actb (Mm02619580_g1), Gfap (Mm01253033_m1), Glud1 (Mm00492353_m1), Glul (Mm00725701_s1), Slc1a3 (Mm00600697_m1), Rbpms (Mm00803908_m1), Mbp (Mm01262037_m1), Iba1 (Mm00479862_g1), Gja1 (Mm00439105_m1).

Techniques: Positive Control, Negative Control

Journal: Evidence-based complementary and alternative medicine : eCAM

Article Title: A Compound of Chinese Herbs Protects against Alcoholic Liver Fibrosis in Rats via the TGF- β 1/Smad Signaling Pathway.

doi: 10.1155/2019/9121347

Figure Lengend Snippet: Figure 11: Protein expression levels of hepatic TGF-𝛽1, p-Smad2, Smad2, p-Smad3, Smad3, and Smad7. Notes: A: blank control group (distilled water); B: model control group (distilled water); C: positive control group (3 g/kg⋅bw/day, positive medicine); D: low dose group (0.333 g/kg⋅bw/day, PSSS compound); E: medium dose group (0.667 g/kg⋅bw/day, PSSS compound); F: High dose group (1 g/kg⋅bw/day, PSSS compound). TGF-𝛽1, transforming growth factor-𝛽1.

Article Snippet: PVDF membranes were blocked in 5% bovine serum albumin dissolved in tris-buffered saline containing Tween20 (TBST) for 2 h and then incubated in primary rabbit antirat TGF-β1 (monoclonal, 1:1000, Abcam, USA), rabbit antirat p-Smad2, rabbit anti-rat Smad2, rabbit anti-rat p-Smad3, rabbit anti-rat Smad3 (all monoclonal, 1:1000, CST, USA), rabbit anti-rat Smad7 (polyclonal, 1:1000, Proteintech group, Wuhan, China), and rabbit anti-rat GADPH (polyclonal, 1:10000, Proteintech group, Wuhan, China) overnight at 4∘C.

Techniques: Expressing, Control, Positive Control

Journal: Carbohydrate polymers

Article Title: Enhanced mechanical properties of bacterial cellulose nanocomposites produced by co-culturing Gluconacetobacter hansenii and Escherichia coli under static conditions.

doi: 10.1016/j.carbpol.2019.04.071

Figure Lengend Snippet: Fig. 1. FESEM images of surface (a) and cross- section (d) of control BC (monoculture of G. hansenii); surface (b) and cross-section (e) of C23769 & 700728 BC (co-culture of G. hansenii and E. coli ATCC 700728); surface (c) and cross-section (f) of C23769 & 35860 BC (co- culture of G. hansenii and E. coli ATCC 35860). The red circles indicate ambiguous boundaries between different BC layers (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Article Snippet: With the presence of EPS7 produced by E. coli ATCC 700728 under co-culture, the microfibrils between the layers were bound together and a part of the porous regions between adjacent layers was filled with EPS.

Techniques: Control, Co-Culture Assay

Journal: Carbohydrate polymers

Article Title: Enhanced mechanical properties of bacterial cellulose nanocomposites produced by co-culturing Gluconacetobacter hansenii and Escherichia coli under static conditions.

doi: 10.1016/j.carbpol.2019.04.071

Figure Lengend Snippet: Fig. 2. Box plots of distance between adjacent layers in cross section FESEM images. The box indicates the percentage between 25% and 75%. The outliner coefficient is equal to 1.5. The line crossed the box shows the average value. Control BC: monoculture of G. hansenii; C23769 & 700728 BC: co-culture of G. hansenii and E. coli ATCC 700728; C23769 & 35860 BC: co-culture of G. hansenii and E. coli ATCC 35860.

Article Snippet: With the presence of EPS7 produced by E. coli ATCC 700728 under co-culture, the microfibrils between the layers were bound together and a part of the porous regions between adjacent layers was filled with EPS.

Techniques: Control, Co-Culture Assay

Journal: Carbohydrate polymers

Article Title: Enhanced mechanical properties of bacterial cellulose nanocomposites produced by co-culturing Gluconacetobacter hansenii and Escherichia coli under static conditions.

doi: 10.1016/j.carbpol.2019.04.071

Figure Lengend Snippet: Fig. 5. Characterization of biological para- meters during co-culturing of G. hansenii ATCC 23769 and E. coli ATCC 700728 over 120 h with the monoculture of the two strains as control groups: (a) glucose concentration in culture media; (b) CFU per milliliter; (c) bio- mass in dry weight; (d) BC pellicles in dry weigh; (n = 3). C23769 & 700728: co-culture of G. hansenii ATCC 23769 and E. coli ATCC 700728, M23769: monoculture of G. hansenii ATCC 23769, M700728: monoculture of E. coli ATCC 700728, C700728: CFU of E. coli ATCC 700728 during co-culturing, C23769: CFU of G. hansenii ATCC 23769 during co-culturing.

Article Snippet: With the presence of EPS7 produced by E. coli ATCC 700728 under co-culture, the microfibrils between the layers were bound together and a part of the porous regions between adjacent layers was filled with EPS.

Techniques: Control, Concentration Assay, Co-Culture Assay

Journal: Carbohydrate polymers

Article Title: Enhanced mechanical properties of bacterial cellulose nanocomposites produced by co-culturing Gluconacetobacter hansenii and Escherichia coli under static conditions.

doi: 10.1016/j.carbpol.2019.04.071

Figure Lengend Snippet: Fig. 6. The EPS concentration produced by E. coli ATCC 700728 during its monoculture (dark columns) and co-culturing (grey columns) with G. hansenii ATCC 23769 (n = 3).

Article Snippet: With the presence of EPS7 produced by E. coli ATCC 700728 under co-culture, the microfibrils between the layers were bound together and a part of the porous regions between adjacent layers was filled with EPS.

Techniques: Concentration Assay, Produced

Journal: Carbohydrate polymers

Article Title: Enhanced mechanical properties of bacterial cellulose nanocomposites produced by co-culturing Gluconacetobacter hansenii and Escherichia coli under static conditions.

doi: 10.1016/j.carbpol.2019.04.071

Figure Lengend Snippet: Fig. 7. Chemical media with different carbon sources for the growth of G. hansenii: (a) without glucose, negative control; (b) with 1.0% (w/v) glucose, positive control; (c) replacing glucose with 1.0% (w/v) EPS from monoculture E. coli ATCC 700728. The red arrow indicates the presence of cellulose (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Article Snippet: With the presence of EPS7 produced by E. coli ATCC 700728 under co-culture, the microfibrils between the layers were bound together and a part of the porous regions between adjacent layers was filled with EPS.

Techniques: Negative Control, Positive Control

Journal: RSC Advances

Article Title: Development of bactericidal spinel ferrite nanoparticles with effective biocompatibility for potential wound healing applications

doi: 10.1039/d0ra08417d

Figure Lengend Snippet: Fluorescent microscopy images of E. coli and S. aureus after treatment with NPs. (A) E. coli without any treatment (negative control), (B) E. coli treated with tetracycline (positives control), (C) E. coli treated with 250 μg ml −1 of NF NPs, (D) E. coli treated with 125 μg ml −1 of ZNF NPs, (E) S. aureus without treatment (negative control), (F) S. aureus treated with tetracycline (positives control), (G) S. aureus treated with 250 μg ml −1 of NF NPs and (H) S. aureus treated with 125 μg ml −1 of ZNF NPs. Percent dead cells of (I) E. coli and (J) S. aureus after treatment with different concentration of NPs. Experiments were performed in triplicate. The percent dead cells of E. coli and S. aureus was significantly higher in NP treated groups and positive control (PC) as compare to negative control (NC), (** p ≤ 0.01, *** p ≤ 0.001).

Article Snippet: All the bacterial strains i.e. Escherichia coli ( E. coli ) (ATCC 15224), Pseudomonas aeruginosa ( P. aeruginosa ) (ATCC-15442), Klebsiella pneumoniae ( K. pneumoniae ) B5055, Salmonella typhi ( S. typhi ) (ATCC 14028) and Staphylococcus aureus ( S. aureus ) (ATCC 6538) were kindly provided by Department of Pharmacy, Quaid-i-Azam University, Pakistan.

Techniques: Microscopy, Negative Control, Control, Concentration Assay, Positive Control

Journal: RSC Advances

Article Title: Development of bactericidal spinel ferrite nanoparticles with effective biocompatibility for potential wound healing applications

doi: 10.1039/d0ra08417d

Figure Lengend Snippet: Fluorescent microscopy images of E. coli and S. aureus showing the influx of FITC after treatment with NPs and tetracycline (positive control). (A) Untreated E. coli (negative control), (B) E. coli treated with tetracycline, (C) E. coli treated with 250 μg ml −1 of NF NPs, (D) E. coli treated with 125 μg ml −1 of ZNF NPs, (E) untreated S. aureus (negative control), (F) S. aureus treated with tetracycline, (G) S. aureus treated with 250 μg ml −1 of NF NPs and (H) S. aureus treated with 125 μg ml −1 of ZNF NPs. Tetracycline, NF and ZNF NPs induced membrane damaged to both selected strains of Gram-positive and Gram-negative bacteria.

Article Snippet: All the bacterial strains i.e. Escherichia coli ( E. coli ) (ATCC 15224), Pseudomonas aeruginosa ( P. aeruginosa ) (ATCC-15442), Klebsiella pneumoniae ( K. pneumoniae ) B5055, Salmonella typhi ( S. typhi ) (ATCC 14028) and Staphylococcus aureus ( S. aureus ) (ATCC 6538) were kindly provided by Department of Pharmacy, Quaid-i-Azam University, Pakistan.

Techniques: Microscopy, Positive Control, Negative Control, Membrane, Bacteria

Journal: RSC Advances

Article Title: Development of bactericidal spinel ferrite nanoparticles with effective biocompatibility for potential wound healing applications

doi: 10.1039/d0ra08417d

Figure Lengend Snippet: The effects of NF and ZNF NPs on protein leakage from (A) E. coli and (B) S. aureus after 8 h of treatment at the mentioned concentrations. All experiment were performed in triplicate and data are presented with ±SD. * p ≤ 0.05, ** p ≤ 0.01, ** p ≤ 0.001 were considered statistically significant.

Article Snippet: All the bacterial strains i.e. Escherichia coli ( E. coli ) (ATCC 15224), Pseudomonas aeruginosa ( P. aeruginosa ) (ATCC-15442), Klebsiella pneumoniae ( K. pneumoniae ) B5055, Salmonella typhi ( S. typhi ) (ATCC 14028) and Staphylococcus aureus ( S. aureus ) (ATCC 6538) were kindly provided by Department of Pharmacy, Quaid-i-Azam University, Pakistan.

Techniques:

Journal: RSC Advances

Article Title: Development of bactericidal spinel ferrite nanoparticles with effective biocompatibility for potential wound healing applications

doi: 10.1039/d0ra08417d

Figure Lengend Snippet: Generation of intracellular ROS by NF and ZNF NPs after 8 h of treatment in (A) E. coli and (B) S. aureus . H 2 O 2 treated cells were taken as positive control (PC) and cells without treatment as negative control (NC). All experiments were performed in triplicates, and data are presented as ±SD. * p ≤ 0.05, ** p ≤ 0.01 and*** p ≤ 0.001 were considered as statistically significant.

Article Snippet: All the bacterial strains i.e. Escherichia coli ( E. coli ) (ATCC 15224), Pseudomonas aeruginosa ( P. aeruginosa ) (ATCC-15442), Klebsiella pneumoniae ( K. pneumoniae ) B5055, Salmonella typhi ( S. typhi ) (ATCC 14028) and Staphylococcus aureus ( S. aureus ) (ATCC 6538) were kindly provided by Department of Pharmacy, Quaid-i-Azam University, Pakistan.

Techniques: Positive Control, Negative 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: 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:

Journal: BMC Genomics

Article Title: Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe

doi: 10.1186/1471-2164-13-161

Figure Lengend Snippet: Lox66/lox71-mediated DNA integration and cloning of insertion mutation. (A) Integration of a bacterial plasmid DNA into the insertion mutation of a S. pombe insertion mutant. The plasmid pLox66 bearing the lox66 sequence can recombine with lox71 on the integrated insertion vector in S. pombe in the presence of Cre recombinase. After pLox66 integration, pLox66, the insertion vector and nearby S. pombe genomic DNA can be excised by restriction digestion and cloned in E. coli . The pUC origin allows pLox66 to be amplified and maintained in E. coli and Kan R /G418 R gene ( kanMX ) allow selection of the plasmid in E. coli (kanamycin resistance) and S. pombe (G418 resistance). (B) Cre recombinase-dependent integration of pLox66 in S. pombe . The pLox66 DNA was transformed to S. pombe insertion mutant strains 18_M24 or that expressed or did not express Cre recombinase (pREP81-Cre or pREP81). Transformed cells were replica plated to solid media with G418 to test G418 resistance and stable integration of pLox66. (C) Stable integration of pLox66 in 18_M24 was tested by PCR using primers on the insertion vector (A and D in panel A) and pLox66 (B and C in panel A). A truncated sck1 + gene fragment was co-amplified in each reaction as a positive control. Five independent colonies of each transformation were tested. (D) PCR products of 18_M24 with pREP81-Cre were sequenced to examine the recombined wild type loxP and lox66/71 hybrid sequences. The colored boxes in the electropherograms highlight the base differences in the individual lox71 and lox66 sites while the black boxes indicate the wild type loxP sequences.

Article Snippet: The E. coli electrocompetent cell NEB 5-alpha (Cat# C2989K, NEB) was used for the construction of the bacterial barcode-tagged insertion DNA library.

Techniques: Clone Assay, Mutagenesis, Plasmid Preparation, Sequencing, Amplification, Selection, Transformation Assay, Positive Control