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Greiner Bio 6 well plates
6 Well Plates, supplied by Greiner Bio, used in various techniques. Bioz Stars score: 92/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/6 well plates/product/Greiner Bio
Average 92 stars, based on 49 article reviews
Price from $9.99 to $1999.99
6 well plates - by Bioz Stars, 2020-09
92/100 stars

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Centrifugation:

Article Title: Establishment and Characterization of an SV40 Large T Antigen-Transduced Porcine Colonic Epithelial Cell Line.
Article Snippet: .. Continuous cell lines have become indispensable tools that have enabled investigations into cellular mechanisms by increasing experimental reproducibility and sample availability, and decreasing the use of experimental animals. .. Continuous cell lines have become indispensable tools that have enabled investigations into cellular mechanisms by increasing experimental reproducibility and sample availability, and decreasing the use of experimental animals.

Multiple Displacement Amplification:

Article Title: Repurposing the antidepressant sertraline as SHMT inhibitor to suppress serine/glycine synthesis addicted breast tumor growth
Article Snippet: .. A number of 150.000 MDA-MB-468 cells were plated in 2 ml of DMEM culture medium (Life Technologies) in 6-well plates (Greiner Bio-One) and incubated for 24 h at 37 °C to obtain optimal adherence to the surface. ..

Transfection:

Article Title: Cross‐typic specificity and immunotherapeutic potential of a human HPV16 E7‐specific CTL line
Article Snippet: .. To carry out transfection, C33A cells were plated out at 2×105 /well in 6‐well plates (Greiner Bio‐One, Ltd., Stonehouse, Gloucestershire, UK) for 18 hr before the addition of DOTAP liposomal transfection reagent (Roche Diagnostics, Ltd., Lewes, East Sussex, UK, according to the manufacturer's instructions) and 5 μg DNA. .. Forty‐eight hours posttransfection E7 expressing cells were selected in 2.5 μg/ml blasticidin (Invitrogen Corporation).

Cell Culture:

Article Title: Novel Role for Animal Innate Immune Molecules: Enterotoxic Activity of a Snail Egg MACPF-Toxin
Article Snippet: .. Caco-2 cells were cultured in 6-well plates (Greiner Bio-One) and incubated at 37°C for 24 h. Then 100 μL of a PBS solution containing 100 μg of PmPV2 was added 24, 12, 3, 1, 0.5, and 0.25 h before trypsinization; 100 μL of PBS buffer was used as control. .. After harvest, cell suspensions were washed two times and resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2 pH 7.4) at 2 × 106 cells/mL.

Article Title: Altering the bilayer motif in ERBB2 HER2 TMD and in ErbB HER TMD dimer causing in vitro and in vivo tumor suppression
Article Snippet: .. Approximately 350,000 cells were cultured in 2 ml of culture medium in a well (6-well plates were used, Greiner, Nurtinger, Germany) and incubated for 24 h with different doses of AKOS004122375 in the absence of fetal calf serum. ..

Article Title: Isothiocyanate E‐4IB induces MAPK activation, delayed cell cycle transition and apoptosis
Article Snippet: .. Cells were plated at 0.5 × 106 cells/ml density and cultured in 96‐, 24‐ or 6‐well plates (Greiner, Germany). .. Cells were synchronized at the G1 /S‐phase boundary using single thymidine block as previously described ( ).

Incubation:

Article Title: Repurposing the antidepressant sertraline as SHMT inhibitor to suppress serine/glycine synthesis addicted breast tumor growth
Article Snippet: .. A number of 150.000 MDA-MB-468 cells were plated in 2 ml of DMEM culture medium (Life Technologies) in 6-well plates (Greiner Bio-One) and incubated for 24 h at 37 °C to obtain optimal adherence to the surface. ..

Article Title: Novel Role for Animal Innate Immune Molecules: Enterotoxic Activity of a Snail Egg MACPF-Toxin
Article Snippet: .. Caco-2 cells were cultured in 6-well plates (Greiner Bio-One) and incubated at 37°C for 24 h. Then 100 μL of a PBS solution containing 100 μg of PmPV2 was added 24, 12, 3, 1, 0.5, and 0.25 h before trypsinization; 100 μL of PBS buffer was used as control. .. After harvest, cell suspensions were washed two times and resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2 pH 7.4) at 2 × 106 cells/mL.

Article Title: Altering the bilayer motif in ERBB2 HER2 TMD and in ErbB HER TMD dimer causing in vitro and in vivo tumor suppression
Article Snippet: .. Approximately 350,000 cells were cultured in 2 ml of culture medium in a well (6-well plates were used, Greiner, Nurtinger, Germany) and incubated for 24 h with different doses of AKOS004122375 in the absence of fetal calf serum. ..

Flow Cytometry:

Article Title: Gold Nanocomplex Strongly Modulates the PI3K/Akt Pathway and Other Pathways in MCF-7 Breast Cancer Cell Line
Article Snippet: .. Flow Cytometry MCF-7 cells (American Type Culture Collection (ATCC), USA) (4 × 105 cells) were seeded into 6-well plates (Greiner Bio-One, Frickenhausen, Germany) until reaching confluency. ..

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  • 88
    Greiner Bio 6 well plate wells
    Vitronectin induces ruffled borders and clear zones in osteoclasts. Osteoclasts were incubated for 5 hours in MEM/BSA with M-CSF (50 ng/ml), RANKL (30 ng/ml) and IL-1α (10 ng/ml) in <t>6-well</t> plate wells coated with vitronectin or fibronectin (50 µg/ml), before raising into suspension with a cell scraper and preparation for TEM. A: Osteoclast incubated on vitronectin shows a central area of ruffled border (arrowhead) and a peripheral area free of organelles (‘clear zone’) (arrows). B, C: higher magnification of center (B) and lower portion (C) respectively of A, showing area of ruffled border (B) and clear zone (C); D–F: Osteoclast incubated on fibronectin shows well-spread appearance, but the undersurface lacked the membrane folds and clear zones seen in osteoclasts incubated on vitronectin. E and F are from central and lower portion of D respectively. Scale bars: A, D: 5 µm; B, C, E, F: 1 µm.
    6 Well Plate Wells, supplied by Greiner Bio, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/6 well plate wells/product/Greiner Bio
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    6 well plate wells - by Bioz Stars, 2020-09
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    85
    Greiner Bio poly l lysine laminin coated 6 well plates
    IPA-3 effect on cell spreading in human primary Schwann cells (NF2+/+) Schwann cells that were allowed to spread for 30 minutes in the absence or presence of the PAK inhibitor IPA-3 on <t>poly-L-lysine/laminin</t> coated dishes were stained for the Arp2/3 complex
    Poly L Lysine Laminin Coated 6 Well Plates, supplied by Greiner Bio, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/poly l lysine laminin coated 6 well plates/product/Greiner Bio
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    92
    Greiner Bio mscs
    Knock-down of IGFBP5 mimics most of the CAF-CM-induced effects on signaling pathways and protein expression A. RT-PCR analysis of IGFBP5 mRNA levels in <t>MCF-7</t> cells transfected with siIGFBP5, siSTAT3 or siLuc (control siRNA) followed by treatment with CAF-CM or no treatment. B. Western blot analyses of levels of stromal cell-regulated proteins and phospho-proteins after treatment of MCF-7 cells with siIGFBP5 or siLuc in the presence or absence of CAF-CM (CE = cytosolic extract, NE = nuclear extract, PM = plasma membrane extract). To check for equal loading of plasma membrane proteins, proteins remaining in the gel after blotting were stained with Coomassie Blue (Coom.) C. Effects of two different human <t>MSCs</t> isolates on IGFBP5 and Bcl-3 levels in MCF-7 cells. Either MSCs were co-cultured with MCF-7 cells in a ratio of 1:50 or 20% MSC-CM was added to the MCF-7 cells. D. RT-PCR analyses of RNAs isolated from MCF-7 cells treated with CAF-CM (•) or from untreated MCF-7 cells (○) for IGFBP5 and Bcl-3 mRNA levels. E, F. Comparison of the Bcl-3 mRNA RNA (E) and protein (F) levels in the presence and absence of CAF-CM in a time course experiment. For each time point, the difference in Bcl-3 expression between control cells and CAF-CM-treated cells is statistically significant as determined by paired sample student's t -test. In (A, D, F), each bar represents the mean value ± S.D. of at least three independent experiments.
    Mscs, supplied by Greiner Bio, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mscs/product/Greiner Bio
    Average 92 stars, based on 9 article reviews
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    90
    Greiner Bio l312s v2r nluc
    Electropherograms of DNA sequences of the <t>AVPR2</t> gene. Hemizygous c.935T > C transition (exon 3) changing leucine (TTG) to serine (TCG) at position 312 <t>(L312S)</t> in the proband (A). Heterozygous L312S mutation in the mother (B). Wild-type sequence (C).
    L312s V2r Nluc, supplied by Greiner Bio, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Vitronectin induces ruffled borders and clear zones in osteoclasts. Osteoclasts were incubated for 5 hours in MEM/BSA with M-CSF (50 ng/ml), RANKL (30 ng/ml) and IL-1α (10 ng/ml) in 6-well plate wells coated with vitronectin or fibronectin (50 µg/ml), before raising into suspension with a cell scraper and preparation for TEM. A: Osteoclast incubated on vitronectin shows a central area of ruffled border (arrowhead) and a peripheral area free of organelles (‘clear zone’) (arrows). B, C: higher magnification of center (B) and lower portion (C) respectively of A, showing area of ruffled border (B) and clear zone (C); D–F: Osteoclast incubated on fibronectin shows well-spread appearance, but the undersurface lacked the membrane folds and clear zones seen in osteoclasts incubated on vitronectin. E and F are from central and lower portion of D respectively. Scale bars: A, D: 5 µm; B, C, E, F: 1 µm.

    Journal: PLoS ONE

    Article Title: Bone Is Not Essential for Osteoclast Activation

    doi: 10.1371/journal.pone.0012837

    Figure Lengend Snippet: Vitronectin induces ruffled borders and clear zones in osteoclasts. Osteoclasts were incubated for 5 hours in MEM/BSA with M-CSF (50 ng/ml), RANKL (30 ng/ml) and IL-1α (10 ng/ml) in 6-well plate wells coated with vitronectin or fibronectin (50 µg/ml), before raising into suspension with a cell scraper and preparation for TEM. A: Osteoclast incubated on vitronectin shows a central area of ruffled border (arrowhead) and a peripheral area free of organelles (‘clear zone’) (arrows). B, C: higher magnification of center (B) and lower portion (C) respectively of A, showing area of ruffled border (B) and clear zone (C); D–F: Osteoclast incubated on fibronectin shows well-spread appearance, but the undersurface lacked the membrane folds and clear zones seen in osteoclasts incubated on vitronectin. E and F are from central and lower portion of D respectively. Scale bars: A, D: 5 µm; B, C, E, F: 1 µm.

    Article Snippet: Flexible silicone rubber films were prepared by adding 25 µl silicon DC-200 to the centre of 6-well plate wells (Greiner Bio-One, Stonehouse, Gloucestershire, UK) and allowing to spread for 18 h. The surface of the silicone was then polymerized by sputter coating with gold (20 mA; 20 seconds).

    Techniques: Incubation, Transmission Electron Microscopy

    IPA-3 effect on cell spreading in human primary Schwann cells (NF2+/+) Schwann cells that were allowed to spread for 30 minutes in the absence or presence of the PAK inhibitor IPA-3 on poly-L-lysine/laminin coated dishes were stained for the Arp2/3 complex

    Journal: Experimental neurology

    Article Title: PAK kinase regulates Rac GTPase and is a potential target in human schwannomas

    doi: 10.1016/j.expneurol.2009.04.019

    Figure Lengend Snippet: IPA-3 effect on cell spreading in human primary Schwann cells (NF2+/+) Schwann cells that were allowed to spread for 30 minutes in the absence or presence of the PAK inhibitor IPA-3 on poly-L-lysine/laminin coated dishes were stained for the Arp2/3 complex

    Article Snippet: Briefly, cells were cultured in proliferation medium and grown on poly-L-lysine/laminin coated 6-well plates (Greiner bio-one, Stonehouse, UK), 8-well permanox chamber slides (Nunc, Wiesbaden, Germany) or glass-bottom-dishes (Mattek, Ashland, MA, USA).

    Techniques: Indirect Immunoperoxidase Assay, Staining

    IPA-3 effect on cell spreading in human primary schwannoma cells (NF2−/−) Schwannoma cells that were allowed to spread for 30 minutes in the absence or presence of the PAK inhibitor IPA-3 or the control substance PIR-3.5 on poly-L-lysine/laminin

    Journal: Experimental neurology

    Article Title: PAK kinase regulates Rac GTPase and is a potential target in human schwannomas

    doi: 10.1016/j.expneurol.2009.04.019

    Figure Lengend Snippet: IPA-3 effect on cell spreading in human primary schwannoma cells (NF2−/−) Schwannoma cells that were allowed to spread for 30 minutes in the absence or presence of the PAK inhibitor IPA-3 or the control substance PIR-3.5 on poly-L-lysine/laminin

    Article Snippet: Briefly, cells were cultured in proliferation medium and grown on poly-L-lysine/laminin coated 6-well plates (Greiner bio-one, Stonehouse, UK), 8-well permanox chamber slides (Nunc, Wiesbaden, Germany) or glass-bottom-dishes (Mattek, Ashland, MA, USA).

    Techniques: Indirect Immunoperoxidase Assay

    Knock-down of IGFBP5 mimics most of the CAF-CM-induced effects on signaling pathways and protein expression A. RT-PCR analysis of IGFBP5 mRNA levels in MCF-7 cells transfected with siIGFBP5, siSTAT3 or siLuc (control siRNA) followed by treatment with CAF-CM or no treatment. B. Western blot analyses of levels of stromal cell-regulated proteins and phospho-proteins after treatment of MCF-7 cells with siIGFBP5 or siLuc in the presence or absence of CAF-CM (CE = cytosolic extract, NE = nuclear extract, PM = plasma membrane extract). To check for equal loading of plasma membrane proteins, proteins remaining in the gel after blotting were stained with Coomassie Blue (Coom.) C. Effects of two different human MSCs isolates on IGFBP5 and Bcl-3 levels in MCF-7 cells. Either MSCs were co-cultured with MCF-7 cells in a ratio of 1:50 or 20% MSC-CM was added to the MCF-7 cells. D. RT-PCR analyses of RNAs isolated from MCF-7 cells treated with CAF-CM (•) or from untreated MCF-7 cells (○) for IGFBP5 and Bcl-3 mRNA levels. E, F. Comparison of the Bcl-3 mRNA RNA (E) and protein (F) levels in the presence and absence of CAF-CM in a time course experiment. For each time point, the difference in Bcl-3 expression between control cells and CAF-CM-treated cells is statistically significant as determined by paired sample student's t -test. In (A, D, F), each bar represents the mean value ± S.D. of at least three independent experiments.

    Journal: Oncotarget

    Article Title: Stromal cells promote anti-estrogen resistance of breast cancer cells through an insulin-like growth factor binding protein 5 (IGFBP5)/B-cell leukemia/lymphoma 3 (Bcl-3) axis

    doi:

    Figure Lengend Snippet: Knock-down of IGFBP5 mimics most of the CAF-CM-induced effects on signaling pathways and protein expression A. RT-PCR analysis of IGFBP5 mRNA levels in MCF-7 cells transfected with siIGFBP5, siSTAT3 or siLuc (control siRNA) followed by treatment with CAF-CM or no treatment. B. Western blot analyses of levels of stromal cell-regulated proteins and phospho-proteins after treatment of MCF-7 cells with siIGFBP5 or siLuc in the presence or absence of CAF-CM (CE = cytosolic extract, NE = nuclear extract, PM = plasma membrane extract). To check for equal loading of plasma membrane proteins, proteins remaining in the gel after blotting were stained with Coomassie Blue (Coom.) C. Effects of two different human MSCs isolates on IGFBP5 and Bcl-3 levels in MCF-7 cells. Either MSCs were co-cultured with MCF-7 cells in a ratio of 1:50 or 20% MSC-CM was added to the MCF-7 cells. D. RT-PCR analyses of RNAs isolated from MCF-7 cells treated with CAF-CM (•) or from untreated MCF-7 cells (○) for IGFBP5 and Bcl-3 mRNA levels. E, F. Comparison of the Bcl-3 mRNA RNA (E) and protein (F) levels in the presence and absence of CAF-CM in a time course experiment. For each time point, the difference in Bcl-3 expression between control cells and CAF-CM-treated cells is statistically significant as determined by paired sample student's t -test. In (A, D, F), each bar represents the mean value ± S.D. of at least three independent experiments.

    Article Snippet: In transwell experiments, MSCs were separated from MCF-7 cells by a 0.4 μm pore membrane (Greiner) with the MCF-7 cells grown on the bottom of the well of a 6-well-plate and the MSCs attached to the upper side of the membrane.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot, Staining, Cell Culture, Isolation

    MSCs and CAFs promote growth of MCF-7 cells in the presence of fulvestrant Effect of 100 nM fulvestrant on A. colony size of MCF-7 cells in the clonogenic assays, B. expression of ERα, Ki67, P-ERK1/2 and P-AKT levels in Western blot analysis, C. spheroid formation and D. RNA expression of mesenchymal markers (ACTA, VIM, FN1) and stem cell markers (CD44, PROCR, ABCG2, ALDH3A1). E, F. Effects of MSCs, MSC-CM and CAF-CM on MCF-7 cell growth in the presence and absence of fulvestrant. Cell growth was either determined by measuring the sizes of individual clones in the clonogenic assay (E) or by the ATP-based growth assay (F). G, H. Effect of CAF-CM on (G) spheroid size in the presence and absence of fulvestrant and (H) on the RNA levels of mesenchymal and stem cell markers in the absence and presence of fulvestrant. In (A, E), the data of a representative experiment are shown, in (D, F–H), each bar represents the mean value ± S.D. of at least three independent experiments. Statistical analysis for the clonogenic assay was performed by the Wilcoxon test (A, E). Other statistical analyses were done by using the student's t -test. ACTA = α-smooth muscle actin, VIM = vimentin, FN1 = fibronectin-1, ABCG2 = ATP binding cassette subfamily G2), ALDH3A1 (aldehyde dehydrogenase 3 family, member A1), NE/CE = nuclear/cytosolic protein extract, RLU = relative light units.

    Journal: Oncotarget

    Article Title: Stromal cells promote anti-estrogen resistance of breast cancer cells through an insulin-like growth factor binding protein 5 (IGFBP5)/B-cell leukemia/lymphoma 3 (Bcl-3) axis

    doi:

    Figure Lengend Snippet: MSCs and CAFs promote growth of MCF-7 cells in the presence of fulvestrant Effect of 100 nM fulvestrant on A. colony size of MCF-7 cells in the clonogenic assays, B. expression of ERα, Ki67, P-ERK1/2 and P-AKT levels in Western blot analysis, C. spheroid formation and D. RNA expression of mesenchymal markers (ACTA, VIM, FN1) and stem cell markers (CD44, PROCR, ABCG2, ALDH3A1). E, F. Effects of MSCs, MSC-CM and CAF-CM on MCF-7 cell growth in the presence and absence of fulvestrant. Cell growth was either determined by measuring the sizes of individual clones in the clonogenic assay (E) or by the ATP-based growth assay (F). G, H. Effect of CAF-CM on (G) spheroid size in the presence and absence of fulvestrant and (H) on the RNA levels of mesenchymal and stem cell markers in the absence and presence of fulvestrant. In (A, E), the data of a representative experiment are shown, in (D, F–H), each bar represents the mean value ± S.D. of at least three independent experiments. Statistical analysis for the clonogenic assay was performed by the Wilcoxon test (A, E). Other statistical analyses were done by using the student's t -test. ACTA = α-smooth muscle actin, VIM = vimentin, FN1 = fibronectin-1, ABCG2 = ATP binding cassette subfamily G2), ALDH3A1 (aldehyde dehydrogenase 3 family, member A1), NE/CE = nuclear/cytosolic protein extract, RLU = relative light units.

    Article Snippet: In transwell experiments, MSCs were separated from MCF-7 cells by a 0.4 μm pore membrane (Greiner) with the MCF-7 cells grown on the bottom of the well of a 6-well-plate and the MSCs attached to the upper side of the membrane.

    Techniques: Expressing, Western Blot, RNA Expression, Clone Assay, Clonogenic Assay, Growth Assay, Binding Assay

    Electropherograms of DNA sequences of the AVPR2 gene. Hemizygous c.935T > C transition (exon 3) changing leucine (TTG) to serine (TCG) at position 312 (L312S) in the proband (A). Heterozygous L312S mutation in the mother (B). Wild-type sequence (C).

    Journal: Molecular Endocrinology

    Article Title: Mutations of Vasopressin Receptor 2 Including Novel L312S Have Differential Effects on Trafficking

    doi: 10.1210/me.2016-1002

    Figure Lengend Snippet: Electropherograms of DNA sequences of the AVPR2 gene. Hemizygous c.935T > C transition (exon 3) changing leucine (TTG) to serine (TCG) at position 312 (L312S) in the proband (A). Heterozygous L312S mutation in the mother (B). Wild-type sequence (C).

    Article Snippet: Briefly, HEK293FT cells were transfected as described above with plasmid DNA containing wild-type V2R/Nluc or L312S V2R/Nluc (500 ng/well of a 6-well plate) and seeded into white 384-well microplates (Greiner) 24 hours before assay.

    Techniques: Mutagenesis, Sequencing

    Effect of NSIAD V2R mutations on β-arrestin2 recruitment assessed with multiple BRET configurations. HEK293FT cells transiently transfected with cDNA coding for wild-type V2R (black circles), R137C V2R (blue triangles), R137L V2R (red squares), or L312S V2R (orange diamonds) C-terminally tagged with Rluc8 (A) or Nluc (B and C) and β-arrestin2/Venus (A and B) or β-arrestin2/HT (C) were used to determine concentration-response curves with AVP (1pM–10μM) for β-arrestin2 recruitment to wild-type or mutant V2Rs after about 30 minutes. Data are expressed as BRET ratio (above wild-type baseline) as described in Materials and Methods, and the associated kinetic data are shown in Supplemental Figure 1 . Data represent mean ± SEM of 5 independent experiments, with curves fitted by nonlinear regression (GraphPad Prism).

    Journal: Molecular Endocrinology

    Article Title: Mutations of Vasopressin Receptor 2 Including Novel L312S Have Differential Effects on Trafficking

    doi: 10.1210/me.2016-1002

    Figure Lengend Snippet: Effect of NSIAD V2R mutations on β-arrestin2 recruitment assessed with multiple BRET configurations. HEK293FT cells transiently transfected with cDNA coding for wild-type V2R (black circles), R137C V2R (blue triangles), R137L V2R (red squares), or L312S V2R (orange diamonds) C-terminally tagged with Rluc8 (A) or Nluc (B and C) and β-arrestin2/Venus (A and B) or β-arrestin2/HT (C) were used to determine concentration-response curves with AVP (1pM–10μM) for β-arrestin2 recruitment to wild-type or mutant V2Rs after about 30 minutes. Data are expressed as BRET ratio (above wild-type baseline) as described in Materials and Methods, and the associated kinetic data are shown in Supplemental Figure 1 . Data represent mean ± SEM of 5 independent experiments, with curves fitted by nonlinear regression (GraphPad Prism).

    Article Snippet: Briefly, HEK293FT cells were transfected as described above with plasmid DNA containing wild-type V2R/Nluc or L312S V2R/Nluc (500 ng/well of a 6-well plate) and seeded into white 384-well microplates (Greiner) 24 hours before assay.

    Techniques: Bioluminescence Resonance Energy Transfer, Transfection, Concentration Assay, Mutagenesis

    Effect of L312S mutation on AVP mediated G protein signaling. Concentration-response curves to AVP for cAMP (A) and IP 1 (B) accumulation were generated using transiently transfected HEK293FT cells expressing either wild-type (black circles) or L312S (red squares) V2R tagged with Nluc. For cAMP accumulation (A), cells were stimulated with AVP (10fM–0.1μM) or forskolin (100μM) for 30 minutes in buffer containing IBMX (500μM). For IP 1 accumulation (B), cells were stimulated with AVP (1pM–10μM) or carbachol (2mM) for 30 minutes in buffer containing LiCl (50mM). Cells were then lysed, and cAMP or IP 1 accumulation was measured by HTRF. Data shown are normalized to the forskolin or carbachol response for cAMP and IP 1 accumulation respectively and expressed as % of the wild-type V2R maximum response to observe constitutive activity. Points represent mean ± SEM of 4 independent experiments (A) and 6 independent experiments (B), and the curve fit is by nonlinear regression. The effect of 1μM inverse agonist Tolvaptan was also assessed (C) in terms of inhibiting constitutive (black and red bars) or ligand-induced (0.1nM AVP; white and blue bars) cAMP accumulation. Observations were made after 30 minutes in a buffer containing IBMX (500μM), in cells expressing either wild-type or L312S V2R tagged with Nluc. Data are expressed as a percentage of the wild-type response to 0.1nM AVP treatment.

    Journal: Molecular Endocrinology

    Article Title: Mutations of Vasopressin Receptor 2 Including Novel L312S Have Differential Effects on Trafficking

    doi: 10.1210/me.2016-1002

    Figure Lengend Snippet: Effect of L312S mutation on AVP mediated G protein signaling. Concentration-response curves to AVP for cAMP (A) and IP 1 (B) accumulation were generated using transiently transfected HEK293FT cells expressing either wild-type (black circles) or L312S (red squares) V2R tagged with Nluc. For cAMP accumulation (A), cells were stimulated with AVP (10fM–0.1μM) or forskolin (100μM) for 30 minutes in buffer containing IBMX (500μM). For IP 1 accumulation (B), cells were stimulated with AVP (1pM–10μM) or carbachol (2mM) for 30 minutes in buffer containing LiCl (50mM). Cells were then lysed, and cAMP or IP 1 accumulation was measured by HTRF. Data shown are normalized to the forskolin or carbachol response for cAMP and IP 1 accumulation respectively and expressed as % of the wild-type V2R maximum response to observe constitutive activity. Points represent mean ± SEM of 4 independent experiments (A) and 6 independent experiments (B), and the curve fit is by nonlinear regression. The effect of 1μM inverse agonist Tolvaptan was also assessed (C) in terms of inhibiting constitutive (black and red bars) or ligand-induced (0.1nM AVP; white and blue bars) cAMP accumulation. Observations were made after 30 minutes in a buffer containing IBMX (500μM), in cells expressing either wild-type or L312S V2R tagged with Nluc. Data are expressed as a percentage of the wild-type response to 0.1nM AVP treatment.

    Article Snippet: Briefly, HEK293FT cells were transfected as described above with plasmid DNA containing wild-type V2R/Nluc or L312S V2R/Nluc (500 ng/well of a 6-well plate) and seeded into white 384-well microplates (Greiner) 24 hours before assay.

    Techniques: Mutagenesis, Concentration Assay, Generated, Transfection, Expressing, Activity Assay

    Profiling the constitutive localization of (A) gain and (B) loss-of-function V2R mutants. HEK293FT cells were transiently transfected with wild-type or mutant V2R/Rluc8 with or without the plasma membrane marker Venus/K-ras or Venus/Rab intracellular markers. Constitutive localization is plotted relative to wild-type V2R (black circles and line = 1) calculated as described in Materials and Methods. Values more than 1 indicate increased relative basal localization, and values less than 1 indicate reduced relative basal localization. A, NSIAD V2R mutations R137C (blue triangles and line), R137L (red squares and line), and L312S (yellow diamonds and broken line) relative to wild-type V2R (black circles and line). B, NDI V2R mutations R137H (blue triangles and line), R181C (red squares and line), and M311V (yellow diamonds and broken line) relative to wild-type V2R (black circles and line). Points represent the mean of 3 independent experiments. Note the same wild-type data were used in the generation of A and B as well as Supplemental Figure 2 .

    Journal: Molecular Endocrinology

    Article Title: Mutations of Vasopressin Receptor 2 Including Novel L312S Have Differential Effects on Trafficking

    doi: 10.1210/me.2016-1002

    Figure Lengend Snippet: Profiling the constitutive localization of (A) gain and (B) loss-of-function V2R mutants. HEK293FT cells were transiently transfected with wild-type or mutant V2R/Rluc8 with or without the plasma membrane marker Venus/K-ras or Venus/Rab intracellular markers. Constitutive localization is plotted relative to wild-type V2R (black circles and line = 1) calculated as described in Materials and Methods. Values more than 1 indicate increased relative basal localization, and values less than 1 indicate reduced relative basal localization. A, NSIAD V2R mutations R137C (blue triangles and line), R137L (red squares and line), and L312S (yellow diamonds and broken line) relative to wild-type V2R (black circles and line). B, NDI V2R mutations R137H (blue triangles and line), R181C (red squares and line), and M311V (yellow diamonds and broken line) relative to wild-type V2R (black circles and line). Points represent the mean of 3 independent experiments. Note the same wild-type data were used in the generation of A and B as well as Supplemental Figure 2 .

    Article Snippet: Briefly, HEK293FT cells were transfected as described above with plasmid DNA containing wild-type V2R/Nluc or L312S V2R/Nluc (500 ng/well of a 6-well plate) and seeded into white 384-well microplates (Greiner) 24 hours before assay.

    Techniques: Transfection, Mutagenesis, Marker

    Kinetic profiling of the trafficking properties of gain-of-function V2R NSIAD mutants. HEK293FT cells were transiently transfected with wild-type V2R (black circles), R137C V2R (blue triangles), R137L V2R (red squares), or L312S V2R (orange diamonds) tagged with Rluc8 and (A) the plasma membrane marker K-ras or one of the subcellular markers (B) Rab1, (C) Rab4, (D) Rab5, (E) Rab6, (F) Rab7, (G) Rab8, (H) Rab9, or (I) Rab11 tagged with Venus. BRET ratio (ligand-vehicle) was calculated as described in Materials and Methods. AVP (1μM) or vehicle was added at t = 0 after establishment of the baseline. Points represent mean ± SEM of 3 independent experiments. Note wild-type data are replicated in Figure 9 and Supplemental Figure 4 .

    Journal: Molecular Endocrinology

    Article Title: Mutations of Vasopressin Receptor 2 Including Novel L312S Have Differential Effects on Trafficking

    doi: 10.1210/me.2016-1002

    Figure Lengend Snippet: Kinetic profiling of the trafficking properties of gain-of-function V2R NSIAD mutants. HEK293FT cells were transiently transfected with wild-type V2R (black circles), R137C V2R (blue triangles), R137L V2R (red squares), or L312S V2R (orange diamonds) tagged with Rluc8 and (A) the plasma membrane marker K-ras or one of the subcellular markers (B) Rab1, (C) Rab4, (D) Rab5, (E) Rab6, (F) Rab7, (G) Rab8, (H) Rab9, or (I) Rab11 tagged with Venus. BRET ratio (ligand-vehicle) was calculated as described in Materials and Methods. AVP (1μM) or vehicle was added at t = 0 after establishment of the baseline. Points represent mean ± SEM of 3 independent experiments. Note wild-type data are replicated in Figure 9 and Supplemental Figure 4 .

    Article Snippet: Briefly, HEK293FT cells were transfected as described above with plasmid DNA containing wild-type V2R/Nluc or L312S V2R/Nluc (500 ng/well of a 6-well plate) and seeded into white 384-well microplates (Greiner) 24 hours before assay.

    Techniques: Transfection, Marker, Bioluminescence Resonance Energy Transfer