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  • 99
    Vector Laboratories streptavidin
    Dynein crosslinks and slides MTs in bundles. ( A ) Schematic overview of the dynein constructs used in this study. The N-terminal tail is shown in gray, the linker in purple, the six numbered AAA+ domains are in light blue and the stalk and MT binding domain are depicted in orange. GFP and GST tags are shown in green and blue, respectively. The Halo tag (DHA, Promega) is shown in red. ( B ) Coomassie brilliant blue stained gels showing purified dynein constructs used in this study. The associated subunits of the brain cytoplasmic dynein complex are labeled; HC–heavy chain, IC–intermediate chain, LIC–light intermediate chain, LC–light chain. Recombinant yeast dynein constructs do not contain associated subunits. Molecular weight markers are indicated. ( C ) MTs incubated in the absence or presence of dynein are visualized by attachment to a <t>streptavidin-coated</t> coverslip via a biotin tag. Brain dynein and GST-Dyn1 331kDa crosslink MTs into large bundles, while the dynein monomer, Dyn1 331kDa does not. Scale bar, 10 µm. ( D ) Cartoon depicting two different mechanisms by which dynein could crosslink MTs, either using its two motor domains or through the tail domain. Alexa-568 and Alexa-488 labeled MTs are crosslinked by dynein. The green MTs are attached to the coverslip through a biotin-streptavidin linkage and perfusion of 1 mM ATP induces sliding between the MTs. ( E and F ) Example of rat ( E ) and GST-Dyn1 331kDa ( F ) dynein-driven sliding of red-labeled MTs within the bundle after 1 mM ATP addition. Arrowhead tracks the sliding MT within the bundle. The time relative to the start is noted in min:s at the bottom of each image. Scale bar, 5 µm. DOI: http://dx.doi.org/10.7554/eLife.00943.003
    Streptavidin, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 1127 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs streptavidin
    Immobilization of scFv5- linker-BCCP constructs and scFv5-linker-AviTag constructs onto <t>streptavidin-coated</t> plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv5-BCCP with no linker for each biological replicate (n = 6). The error bars represent the standard error of the mean.
    Streptavidin, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 352 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega streptavidin
    (A–B) Representative fluorescence micrographs of glass substrates coated with PEI/PVDMA films spotted with (A) FITC-labeled <t>streptavidin</t> or (B) unlabeled streptavidin (the image in (B) was treated with anti-HA-biotin and anti-rat IgG Alexa Fluor 488 prior to imaging; see text). (C) Digital photograph of film-coated glass containing spots of immobilized β-galactosidase (right spot) or BSA (left spot) incubated under droplets of ONPG for 10 minutes. (D) Plot of ONP concentration vs. time measured from a droplet incubated on a spot containing immobilized β-galactosidase (black squares) or BSA (grey diamonds). Scale bars are (A–B) 1 mm and (C) 2 mm.
    Streptavidin, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 338 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    BioLegend pe streptavidin
    Flow cytometry competition analysis with increasing concentration of the unlabeled (A) or biotinylated (B) foldback diabody anti‐human CCR4 immunotoxins to block the binding of the saturating concentration of the two anti‐human CCR4 mAbs (clone#205410 and L291H4) to the monkey CCR4+ PBMC. Monkey PBMC was incubated with the unlabeled (A) or biotinylated (B) foldback diabody anti‐human immunotoxin at 4 °C for 1 h and washed twice, then stained with the anti‐human CCR4 mAb (A) or the anti‐human CCR4 mAb and <t>APC‐streptavidin</t> (B) at 4 °C for 30 min. The data are representative of two individual experiments.
    Pe Streptavidin, supplied by BioLegend, used in various techniques. Bioz Stars score: 98/100, based on 272 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    BioLegend streptavidin
    Defective 4-1BB activity in T cells and DCs from Gal-9 −/− mice. (a) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD8 + T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (b) 4-1BB and OX40 surface expression on WT and Gal-9 −/− CD8 + T cells activated as in panel a. Mean fluorescent intensity indicated for 4-1BB. Shade, isotype control. (c) Binding of anti–4-1BB (clone 3H3) to activated CD8 + T cells. Activated CD8 + T cells as in panel a were sorted for identical expression of 4-1BB using biotin-anti–4-1BB (Syrian hamster IgG; clone 17B5) and <t>streptavidin-APC.</t> Cells were then stained with unlabeled anti–4-1BB (rat IgG; clone 3H3) and anti–rat IgG FITC to detect anti–4-1BB (clone 3H3) binding to cells. Shade, isotype controls. Data are representative of two different experiments. (d) IFN-γ production by preactivated CD8 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (postsort FACS plot shown) and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of at least three different experiments. (e and f) CD8 + T cells from WT or Gal-9 −/− mice were activated as in panel a, sorted for identical expression of 4-1BB, and recultured for another 24 h to assay IFN-γ production in response to irradiated 4-1BBL + hybridoma cells (e) or varying concentrations of anti-polyhistidine cross-linked r4-1BBL (f). Neutralizing anti–4-1BBL or rat IgG was added to cultures in e. Data are means ± SEM from triplicate cultures and representative of two different experiments. (g, left) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD4 T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (right) IL-2 production by preactivated CD4 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (pre- and postsort FACS plot shown), and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of three different experiments. (h) Surface expression of 4-1BB and Gal-9 (top) and 4-1BB and MHC II (bottom) on ex vivo WT mesenteric LN DCs and splenic DCs from WT and Gal-9 −/− mice cultured with GM-CSF, respectively. Shade, isotype control. (i) ALDEFLUOR staining in preactivated spleen DCs from WT and Gal-9 −/− mice, sorted for identical expression of 4-1BB, and recultured for 24 h in the presence of zymosan with control rat IgG (left) or agonist anti–4-1BB (right). Numbers indicate percentage of cells in each quadrant. Data are representative of three different experiments.
    Streptavidin, supplied by BioLegend, used in various techniques. Bioz Stars score: 95/100, based on 390 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    BioLegend streptavidin apc
    Generation of cTag8-Expressing 293T Cells for Modified Vector Production (A) Both 293T (non-transduced) and 293T cells expressing cTag8 (cTag8 293T) by γ-retroviral transduction with cTag8 co-expressed with EGFP were stained with <t>streptavidin-APC</t> for cTag8 expression analysis by flow cytometry. (B) Surface expression analysis of cTag8 by immunofluorescence staining of cTag8 293T cells with streptavidin-APC. Engineered cells were assessed for LV packaging capacity by the production of LVs from both 293T cells (non-modified, NM LVs) and cTag8 293T cells (cTag8 LVs) in plain DMEM, pseudotyped with either RDpro, MLV-ampho, or VSV-G glycoproteins. (C) Viral supernatants were frozen, viral titers (infectious units (IU)/mL) of stocks were determined by infectivity assay, and mean values are presented ± SD of triplicate determinations. (D) Sucrose-cushion-ultracentrifuge-purified VSV-G pseudotyped NM LVs and cTag8 LVs were negatively stained and analyzed by transmission electron microscopy (TEM). Scale bars, 200 nm.
    Streptavidin Apc, supplied by BioLegend, used in various techniques. Bioz Stars score: 98/100, based on 603 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    BioLegend hrp streptavidin
    Generation of cTag8-Expressing 293T Cells for Modified Vector Production (A) Both 293T (non-transduced) and 293T cells expressing cTag8 (cTag8 293T) by γ-retroviral transduction with cTag8 co-expressed with EGFP were stained with <t>streptavidin-APC</t> for cTag8 expression analysis by flow cytometry. (B) Surface expression analysis of cTag8 by immunofluorescence staining of cTag8 293T cells with streptavidin-APC. Engineered cells were assessed for LV packaging capacity by the production of LVs from both 293T cells (non-modified, NM LVs) and cTag8 293T cells (cTag8 LVs) in plain DMEM, pseudotyped with either RDpro, MLV-ampho, or VSV-G glycoproteins. (C) Viral supernatants were frozen, viral titers (infectious units (IU)/mL) of stocks were determined by infectivity assay, and mean values are presented ± SD of triplicate determinations. (D) Sucrose-cushion-ultracentrifuge-purified VSV-G pseudotyped NM LVs and cTag8 LVs were negatively stained and analyzed by transmission electron microscopy (TEM). Scale bars, 200 nm.
    Hrp Streptavidin, supplied by BioLegend, used in various techniques. Bioz Stars score: 96/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam strepavidin hrp
    SipA binds polymyxin B and LL-37 in vitro and in vivo . Wells from a 96-well plate were coated with either purified SipA or VC1638 (A,B) . LL-37 (A) or biotin-labeled polymyxin B (B) were added to plates and incubated for 1 h. Levels of binding were detected via (A) anti-LL-37 antibody followed by <t>IgG-HRP</t> or (B) <t>Streptavidin-HRP.</t> VC1638 served as a control protein. (C) Coimmunoprecipitation of LL-37 in the presence and absence of 6×His- sipA . Each culture was crosslinked and lysed before loading onto a Ni-NTA column (input), followed by elution with imidazole (output). Blots were probed with anti-LL-37 antibody. Densitometry of the LL-37 band was calculated based on three separate experiments and normalized to the strain containing empty vector. ∗ p =
    Strepavidin Hrp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin antibody
    SipA binds polymyxin B and LL-37 in vitro and in vivo . Wells from a 96-well plate were coated with either purified SipA or VC1638 (A,B) . LL-37 (A) or biotin-labeled polymyxin B (B) were added to plates and incubated for 1 h. Levels of binding were detected via (A) anti-LL-37 antibody followed by <t>IgG-HRP</t> or (B) <t>Streptavidin-HRP.</t> VC1638 served as a control protein. (C) Coimmunoprecipitation of LL-37 in the presence and absence of 6×His- sipA . Each culture was crosslinked and lysed before loading onto a Ni-NTA column (input), followed by elution with imidazole (output). Blots were probed with anti-LL-37 antibody. Densitometry of the LL-37 band was calculated based on three separate experiments and normalized to the strain containing empty vector. ∗ p =
    Streptavidin Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher strepavidin hrp
    Imetelstat (GRN163L) is a competitive inhibitor of primer-substrate binding by telomerase. (A) Experimental design of single-molecule telomerase primer binding and activity assay. Halo-telomerase is modified with a biotin-HaloTag-ligand and immobilized on the coverslip surface using NeutrAvidin. Primer binding is visualized by telomerase-dependent recruitment of a fluorescent primer to the coverslip surface. The telomerase extension product is detected using a fluorescently labeled oligonucleotide anti-sense to the telomerase extension product. (B) Western blot and fluorescence imaging of Halo-telomerase modified with a fluorescent dye (JF646) or biotin, probed with an anti-TERT antibody or <t>HRP-conjugated</t> <t>streptavidin.</t> (C) Single-molecule TIRF imaging of primer molecules recruited to the coverslip surface by telomerase (top) and its colocalization with telomerase extension products after incubation with nucleotide substrate (bottom). (D) Single-molecule TIRF imaging of primer binding by telomerase in the presence of increasing concentrations of imetelstat. (E) Quantification of primer binding to telomerase as a function of imetelstat concentration ( n = 5 fields of view per concentration, data points plotted as mean ± SD, error on IC 50 reflects error in the corresponding fit of the data to a simple binding curve). (F) Direct telomerase assay at 150 mM KCl in the absence and presence of imetelstat (10 nM), or mismatched control oligonucleotide (MM Control, 10 nM), and increasing concentrations of primer substrate. LC1, LC2, and LC3, labeled DNA loading controls. (G) Quantification of telomerase activity as a function of primer concentration in absence and presence of imetelstat (10 nM) or mismatched control oligonucleotide (MM Control, 10 nM).
    Strepavidin Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher streptavidin pe
    Effect of two SpA derivatives on the binding to coated hIgMs and on the T cell presentation of toxin α. (A) Mα2-3 was incubated overnight in the presence or absence of fixed concentrations of toxin α biotinylated at the NH 2 terminus (Alphabiot) and either ZZ or BB in BSA-coated microwell plates. The solutions were transferred in hIgM-coated plates. Binding of Mα2-3 to the wells was determined using a goat anti–mouse IgG peroxidase conjugate (GAM–PO), whereas binding of biotinylated toxin α was determined using a <t>streptavidin</t> peroxidase conjugate (SA–PO). (B) Toxin α (alpha) was serially diluted and incubated overnight at 4°C in the presence or absence of mAb Mα2-3 (25 nM final) and either ZZ or BB (0.1 μM final for each derivative). 5 × 10 5 splenocytes from BALB/c mice were then added to each well in the presence of 5 × 10 4 T1B2. T cell stimulation was assessed as previously described.
    Streptavidin Pe, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2491 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin pe cy7
    A MACS selection procedure for the prospective isolation of progenitor cells for beige/brite differentiation . (A–F) Single cell suspensions were obtained by collagenase digestion of subcutaneous fat, and stained for six-color FACS with the indicated antibodies after removal of adipocytes by centrifugation and erythrocytes by TER119-MACS ® depletion. Debris and singlets were excluded and selected through FSC/SSC and FSC-A/H, respectively. (A–C) and (D–F) represent independent gating schemes. Values (%) indicate cells % of parent plot. Representative plots from multiple independent experiments are shown. (G) Erythrocytes, leukocytes, and endothelial cells were removed from single cell suspensions in a first MACS step with biotinylated Ter119, CD45, and CD31 antibodies and <t>streptavidin-conjugated</t> microbeads. Sca-1 + cells were enriched in a second MACS step with <t>Sca-1-PE-Cy7</t> antibody and anti-PE-Cy7 microbeads. The resulting cell population (Lin − Sca-1 + eluate) as well as the Lin − flow-through were subjected to flow cytometry. Comparable purities were obtained with directly conjugated antibody-bead combinations (data not shown). (H) MACS- and FACS-purified cells were cultured and differentiated for 8 days in the presence or absence of cPGI 2 (see Materials and Methods ) before subjected to RNA expression analysis by qRT-PCR (* indicates t -test p
    Streptavidin Pe Cy7, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    BioLegend strepavidin fitc
    A MACS selection procedure for the prospective isolation of progenitor cells for beige/brite differentiation . (A–F) Single cell suspensions were obtained by collagenase digestion of subcutaneous fat, and stained for six-color FACS with the indicated antibodies after removal of adipocytes by centrifugation and erythrocytes by TER119-MACS ® depletion. Debris and singlets were excluded and selected through FSC/SSC and FSC-A/H, respectively. (A–C) and (D–F) represent independent gating schemes. Values (%) indicate cells % of parent plot. Representative plots from multiple independent experiments are shown. (G) Erythrocytes, leukocytes, and endothelial cells were removed from single cell suspensions in a first MACS step with biotinylated Ter119, CD45, and CD31 antibodies and <t>streptavidin-conjugated</t> microbeads. Sca-1 + cells were enriched in a second MACS step with <t>Sca-1-PE-Cy7</t> antibody and anti-PE-Cy7 microbeads. The resulting cell population (Lin − Sca-1 + eluate) as well as the Lin − flow-through were subjected to flow cytometry. Comparable purities were obtained with directly conjugated antibody-bead combinations (data not shown). (H) MACS- and FACS-purified cells were cultured and differentiated for 8 days in the presence or absence of cPGI 2 (see Materials and Methods ) before subjected to RNA expression analysis by qRT-PCR (* indicates t -test p
    Strepavidin Fitc, supplied by BioLegend, used in various techniques. Bioz Stars score: 95/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore strepavidin phycoerythrin
    A MACS selection procedure for the prospective isolation of progenitor cells for beige/brite differentiation . (A–F) Single cell suspensions were obtained by collagenase digestion of subcutaneous fat, and stained for six-color FACS with the indicated antibodies after removal of adipocytes by centrifugation and erythrocytes by TER119-MACS ® depletion. Debris and singlets were excluded and selected through FSC/SSC and FSC-A/H, respectively. (A–C) and (D–F) represent independent gating schemes. Values (%) indicate cells % of parent plot. Representative plots from multiple independent experiments are shown. (G) Erythrocytes, leukocytes, and endothelial cells were removed from single cell suspensions in a first MACS step with biotinylated Ter119, CD45, and CD31 antibodies and <t>streptavidin-conjugated</t> microbeads. Sca-1 + cells were enriched in a second MACS step with <t>Sca-1-PE-Cy7</t> antibody and anti-PE-Cy7 microbeads. The resulting cell population (Lin − Sca-1 + eluate) as well as the Lin − flow-through were subjected to flow cytometry. Comparable purities were obtained with directly conjugated antibody-bead combinations (data not shown). (H) MACS- and FACS-purified cells were cultured and differentiated for 8 days in the presence or absence of cPGI 2 (see Materials and Methods ) before subjected to RNA expression analysis by qRT-PCR (* indicates t -test p
    Strepavidin Phycoerythrin, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore atto550 strepavidin
    A MACS selection procedure for the prospective isolation of progenitor cells for beige/brite differentiation . (A–F) Single cell suspensions were obtained by collagenase digestion of subcutaneous fat, and stained for six-color FACS with the indicated antibodies after removal of adipocytes by centrifugation and erythrocytes by TER119-MACS ® depletion. Debris and singlets were excluded and selected through FSC/SSC and FSC-A/H, respectively. (A–C) and (D–F) represent independent gating schemes. Values (%) indicate cells % of parent plot. Representative plots from multiple independent experiments are shown. (G) Erythrocytes, leukocytes, and endothelial cells were removed from single cell suspensions in a first MACS step with biotinylated Ter119, CD45, and CD31 antibodies and <t>streptavidin-conjugated</t> microbeads. Sca-1 + cells were enriched in a second MACS step with <t>Sca-1-PE-Cy7</t> antibody and anti-PE-Cy7 microbeads. The resulting cell population (Lin − Sca-1 + eluate) as well as the Lin − flow-through were subjected to flow cytometry. Comparable purities were obtained with directly conjugated antibody-bead combinations (data not shown). (H) MACS- and FACS-purified cells were cultured and differentiated for 8 days in the presence or absence of cPGI 2 (see Materials and Methods ) before subjected to RNA expression analysis by qRT-PCR (* indicates t -test p
    Atto550 Strepavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Dynein crosslinks and slides MTs in bundles. ( A ) Schematic overview of the dynein constructs used in this study. The N-terminal tail is shown in gray, the linker in purple, the six numbered AAA+ domains are in light blue and the stalk and MT binding domain are depicted in orange. GFP and GST tags are shown in green and blue, respectively. The Halo tag (DHA, Promega) is shown in red. ( B ) Coomassie brilliant blue stained gels showing purified dynein constructs used in this study. The associated subunits of the brain cytoplasmic dynein complex are labeled; HC–heavy chain, IC–intermediate chain, LIC–light intermediate chain, LC–light chain. Recombinant yeast dynein constructs do not contain associated subunits. Molecular weight markers are indicated. ( C ) MTs incubated in the absence or presence of dynein are visualized by attachment to a streptavidin-coated coverslip via a biotin tag. Brain dynein and GST-Dyn1 331kDa crosslink MTs into large bundles, while the dynein monomer, Dyn1 331kDa does not. Scale bar, 10 µm. ( D ) Cartoon depicting two different mechanisms by which dynein could crosslink MTs, either using its two motor domains or through the tail domain. Alexa-568 and Alexa-488 labeled MTs are crosslinked by dynein. The green MTs are attached to the coverslip through a biotin-streptavidin linkage and perfusion of 1 mM ATP induces sliding between the MTs. ( E and F ) Example of rat ( E ) and GST-Dyn1 331kDa ( F ) dynein-driven sliding of red-labeled MTs within the bundle after 1 mM ATP addition. Arrowhead tracks the sliding MT within the bundle. The time relative to the start is noted in min:s at the bottom of each image. Scale bar, 5 µm. DOI: http://dx.doi.org/10.7554/eLife.00943.003

    Journal: eLife

    Article Title: Cytoplasmic dynein crosslinks and slides anti-parallel microtubules using its two motor domains

    doi: 10.7554/eLife.00943

    Figure Lengend Snippet: Dynein crosslinks and slides MTs in bundles. ( A ) Schematic overview of the dynein constructs used in this study. The N-terminal tail is shown in gray, the linker in purple, the six numbered AAA+ domains are in light blue and the stalk and MT binding domain are depicted in orange. GFP and GST tags are shown in green and blue, respectively. The Halo tag (DHA, Promega) is shown in red. ( B ) Coomassie brilliant blue stained gels showing purified dynein constructs used in this study. The associated subunits of the brain cytoplasmic dynein complex are labeled; HC–heavy chain, IC–intermediate chain, LIC–light intermediate chain, LC–light chain. Recombinant yeast dynein constructs do not contain associated subunits. Molecular weight markers are indicated. ( C ) MTs incubated in the absence or presence of dynein are visualized by attachment to a streptavidin-coated coverslip via a biotin tag. Brain dynein and GST-Dyn1 331kDa crosslink MTs into large bundles, while the dynein monomer, Dyn1 331kDa does not. Scale bar, 10 µm. ( D ) Cartoon depicting two different mechanisms by which dynein could crosslink MTs, either using its two motor domains or through the tail domain. Alexa-568 and Alexa-488 labeled MTs are crosslinked by dynein. The green MTs are attached to the coverslip through a biotin-streptavidin linkage and perfusion of 1 mM ATP induces sliding between the MTs. ( E and F ) Example of rat ( E ) and GST-Dyn1 331kDa ( F ) dynein-driven sliding of red-labeled MTs within the bundle after 1 mM ATP addition. Arrowhead tracks the sliding MT within the bundle. The time relative to the start is noted in min:s at the bottom of each image. Scale bar, 5 µm. DOI: http://dx.doi.org/10.7554/eLife.00943.003

    Article Snippet: The chamber was coated sequentially with the following solutions: 5 mg/ml BSA-biotin (Sigma, St. Louis, MO), 60 µl BC buffer (BRB80, 1 mg/ml BSA, 1 mg/ml casein, 0.5% Pluronic F-168, pH 6.8), 20 µl 0.5 mg/ml streptavidin (Vector Labs, Burlingame, CA), and 60 µl BC buffer to remove excess streptavidin.

    Techniques: Construct, Binding Assay, Staining, Purification, Labeling, Recombinant, Molecular Weight, Incubation

    Immobilization of scFv5- linker-BCCP constructs and scFv5-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv5-BCCP with no linker for each biological replicate (n = 6). The error bars represent the standard error of the mean.

    Journal: Biotechnology and applied biochemistry

    Article Title: Effect of linkers on immobilization of scFvs with biotin-streptavidin interaction

    doi: 10.1002/bab.1645

    Figure Lengend Snippet: Immobilization of scFv5- linker-BCCP constructs and scFv5-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv5-BCCP with no linker for each biological replicate (n = 6). The error bars represent the standard error of the mean.

    Article Snippet: High-binding 96-well polystyrene plates (Corning) were coated with streptavidin (NEB), as previously described [ ].

    Techniques: Construct, Concentration Assay

    Immobilization of scFv13R4-linker-BCCP constructs and scFv13R4-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv13R4-BCCP with no linker for each biological replicate (n = 5). The error bars represent the standard error of the mean.

    Journal: Biotechnology and applied biochemistry

    Article Title: Effect of linkers on immobilization of scFvs with biotin-streptavidin interaction

    doi: 10.1002/bab.1645

    Figure Lengend Snippet: Immobilization of scFv13R4-linker-BCCP constructs and scFv13R4-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv13R4-BCCP with no linker for each biological replicate (n = 5). The error bars represent the standard error of the mean.

    Article Snippet: High-binding 96-well polystyrene plates (Corning) were coated with streptavidin (NEB), as previously described [ ].

    Techniques: Construct, Concentration Assay

    (A–B) Representative fluorescence micrographs of glass substrates coated with PEI/PVDMA films spotted with (A) FITC-labeled streptavidin or (B) unlabeled streptavidin (the image in (B) was treated with anti-HA-biotin and anti-rat IgG Alexa Fluor 488 prior to imaging; see text). (C) Digital photograph of film-coated glass containing spots of immobilized β-galactosidase (right spot) or BSA (left spot) incubated under droplets of ONPG for 10 minutes. (D) Plot of ONP concentration vs. time measured from a droplet incubated on a spot containing immobilized β-galactosidase (black squares) or BSA (grey diamonds). Scale bars are (A–B) 1 mm and (C) 2 mm.

    Journal: ACS applied materials & interfaces

    Article Title: Fabrication of Oligonucleotide and Protein Arrays on Rigid and Flexible Substrates Coated with Reactive Polymer Multilayers

    doi: 10.1021/am302285n

    Figure Lengend Snippet: (A–B) Representative fluorescence micrographs of glass substrates coated with PEI/PVDMA films spotted with (A) FITC-labeled streptavidin or (B) unlabeled streptavidin (the image in (B) was treated with anti-HA-biotin and anti-rat IgG Alexa Fluor 488 prior to imaging; see text). (C) Digital photograph of film-coated glass containing spots of immobilized β-galactosidase (right spot) or BSA (left spot) incubated under droplets of ONPG for 10 minutes. (D) Plot of ONP concentration vs. time measured from a droplet incubated on a spot containing immobilized β-galactosidase (black squares) or BSA (grey diamonds). Scale bars are (A–B) 1 mm and (C) 2 mm.

    Article Snippet: Streptavidin was purchased from Promega (Madison, WI). β-Galactosidase (β-Gal) was purchased from Prozyme (Hayward, CA).

    Techniques: Fluorescence, Labeling, Imaging, Incubation, Concentration Assay

    Native C. elegans microRNPs contain ALG-1/ALG-2 and bind to complementary targets. ( A ) A biotinylated control DNA sequence, or a mixture of biotinylated DNA oligos perfectly complementary to four abundant miRNAs (mir-58, mir-52, mir-66, mir-71) were bound to streptavidin beads and incubated with enriched miRNPs from N2 worms. Northern blots probed with mir-58, mir-52, mir-66, mir-71 and negative control lin-4 show the enrichment. Silver stain of SDS–PAGE shows co-purified proteins. ALG-1 and ALG-2 were identified by mass spectrometry. ( B ) An in vitro transcribed control RNA and an imperfectly complementary synthetic RNA target for mir-58 were covalently immobilized to beads. RNA-bound beads and a beads-only control were incubated with enriched miRNP fractions from an ALG-1::GFP expressing strain, WM84. The enrichment of mir-58 was detected by northern blot; ALG-1::GFP and TSN-1 were detected by immunoblots.

    Journal: Nucleic Acids Research

    Article Title: The conformation of microRNA seed regions in native microRNPs is prearranged for presentation to mRNA targets

    doi: 10.1093/nar/gkr077

    Figure Lengend Snippet: Native C. elegans microRNPs contain ALG-1/ALG-2 and bind to complementary targets. ( A ) A biotinylated control DNA sequence, or a mixture of biotinylated DNA oligos perfectly complementary to four abundant miRNAs (mir-58, mir-52, mir-66, mir-71) were bound to streptavidin beads and incubated with enriched miRNPs from N2 worms. Northern blots probed with mir-58, mir-52, mir-66, mir-71 and negative control lin-4 show the enrichment. Silver stain of SDS–PAGE shows co-purified proteins. ALG-1 and ALG-2 were identified by mass spectrometry. ( B ) An in vitro transcribed control RNA and an imperfectly complementary synthetic RNA target for mir-58 were covalently immobilized to beads. RNA-bound beads and a beads-only control were incubated with enriched miRNP fractions from an ALG-1::GFP expressing strain, WM84. The enrichment of mir-58 was detected by northern blot; ALG-1::GFP and TSN-1 were detected by immunoblots.

    Article Snippet: The oligonucleotides were immobilized on streptavidin beads and used to specifically enrich their miRNP protein partners.

    Techniques: Sequencing, Incubation, Northern Blot, Negative Control, Silver Staining, SDS Page, Purification, Mass Spectrometry, In Vitro, Expressing, Western Blot

    ( A ) Selection scheme. A pool of mutant ribosomes is used in an in vitro translation reaction where biotin-Phe-tRNA Phe is used as the “initiator” tRNA. Only variants active under the chosen reaction conditions will extend a polypeptide beyond the length of the ribosome's exit channel. Binding to a streptavidin affinity support allows for the isolation of functional ribosomes as peptide-ribosome-mRNA fusions. ( B ) Denaturing PAGE analysis of rRNA recovered after in vitro selection. Translation of a poly(U) mRNA by wild-type (MRE600) ribosomes was initiated with either biotin-Phe-tRNA Phe or N- Ac-Phe-tRNA Phe by using as substrates Phe-tRNA Phe only (lanes 1-3) or both Phe-tRNA Phe and Ser-tRNA SerGAA in a 1:10 ratio (lanes 4-6). Dependence of the reaction on poly(U) mRNA was tested (lanes 2 and 5). The positions of 16S and 23S rRNAs are shown.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Isolation of antibiotic resistance mutations in the rRNA by using an in vitro selection system

    doi: 10.1073/pnas.0307596101

    Figure Lengend Snippet: ( A ) Selection scheme. A pool of mutant ribosomes is used in an in vitro translation reaction where biotin-Phe-tRNA Phe is used as the “initiator” tRNA. Only variants active under the chosen reaction conditions will extend a polypeptide beyond the length of the ribosome's exit channel. Binding to a streptavidin affinity support allows for the isolation of functional ribosomes as peptide-ribosome-mRNA fusions. ( B ) Denaturing PAGE analysis of rRNA recovered after in vitro selection. Translation of a poly(U) mRNA by wild-type (MRE600) ribosomes was initiated with either biotin-Phe-tRNA Phe or N- Ac-Phe-tRNA Phe by using as substrates Phe-tRNA Phe only (lanes 1-3) or both Phe-tRNA Phe and Ser-tRNA SerGAA in a 1:10 ratio (lanes 4-6). Dependence of the reaction on poly(U) mRNA was tested (lanes 2 and 5). The positions of 16S and 23S rRNAs are shown.

    Article Snippet: About 50% of the 10-20% of ribosomes that are active in this modified in vitro translation system were recovered from the streptavidin matrix (i.e., a total of 5-10% of the ribosomal input).

    Techniques: Selection, Mutagenesis, In Vitro, Binding Assay, Isolation, Functional Assay, Polyacrylamide Gel Electrophoresis

    EBP1 binds the CAG repeat sequence in AR codon 1. ( A ) His- EBP1 (150 ng total) was incubated with equimolar amounts of biotinylated CAG repeats of the indicated sizes. The complexes were trapped on magnetic streptavidin beads and associated proteins detected by western blotting. The Bcl-2 UTR was used as a positive control. ( B ) A16 or C13 (EBP1-silenced) cell lysates were incubated with the biotinylated CAG20 repeat or the biotinylated Bcl-2 3′ UTR. Associated proteins were determined by biotin pull-down followed by western blotting. ( C ) A16 or C13 lysates were incubated with the biotinylated GAPDH 3′UTR (GAPDH) or the AR CAG20 repeat. Associated proteins were determined by biotin pull-down followed by western blotting.

    Journal: Nucleic Acids Research

    Article Title: Post-transcriptional regulation of androgen receptor mRNA by an ErbB3 binding protein 1 in prostate cancer

    doi: 10.1093/nar/gkq084

    Figure Lengend Snippet: EBP1 binds the CAG repeat sequence in AR codon 1. ( A ) His- EBP1 (150 ng total) was incubated with equimolar amounts of biotinylated CAG repeats of the indicated sizes. The complexes were trapped on magnetic streptavidin beads and associated proteins detected by western blotting. The Bcl-2 UTR was used as a positive control. ( B ) A16 or C13 (EBP1-silenced) cell lysates were incubated with the biotinylated CAG20 repeat or the biotinylated Bcl-2 3′ UTR. Associated proteins were determined by biotin pull-down followed by western blotting. ( C ) A16 or C13 lysates were incubated with the biotinylated GAPDH 3′UTR (GAPDH) or the AR CAG20 repeat. Associated proteins were determined by biotin pull-down followed by western blotting.

    Article Snippet: Complexes were isolated with paramagnetic streptavidin particles (Promega) and bound proteins were assayed by western blotting using antibodies recognizing EBP1 or GST (Santa Cruz).

    Techniques: Sequencing, Incubation, Western Blot, Positive Control

    In vitro protein‐binding assay of Papaya ringspot virus helper component‐proteinase (PRSV HC‐Pro) and Carica papaya calreticulin (PaCRT). In vitro translated biotinylated PaCRT was incubated with purified glutathione S ‐transferase (GST) or GST‐PRSV HC‐Pro, which was immobilized on MagneGST particles. After incubation, the proteins interacting with GST or GST‐PRSV HC‐Pro were bound to the MagneGST particles. The eluted GST‐bound proteins (lane 4) or GST‐PRSV HC‐Pro‐bound proteins (lane 5) were then analysed by 10% sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and probed with streptavidin‐alkaline phosphatase (streptavidin‐AP) (A) or anti‐GST antibody (B) (GST, 26.0 kDa; GST‐PRSV HC‐Pro, 78.0 kDa; biotinylated PaCRT, 48.2 kDa). The purified GST (lane 1), purified GST‐PRSV HC‐Pro (lane 2) and in vitro translated biotinylated PaCRT (lane 3) were used as controls, respectively. The prestained dual colour protein size marker (Tiangen) is indicated on the left of the panel.

    Journal: Molecular Plant Pathology

    Article Title: Helper component‐proteinase (HC‐Pro) protein of Papaya ringspot virus interacts with papaya calreticulin

    doi: 10.1111/j.1364-3703.2009.00606.x

    Figure Lengend Snippet: In vitro protein‐binding assay of Papaya ringspot virus helper component‐proteinase (PRSV HC‐Pro) and Carica papaya calreticulin (PaCRT). In vitro translated biotinylated PaCRT was incubated with purified glutathione S ‐transferase (GST) or GST‐PRSV HC‐Pro, which was immobilized on MagneGST particles. After incubation, the proteins interacting with GST or GST‐PRSV HC‐Pro were bound to the MagneGST particles. The eluted GST‐bound proteins (lane 4) or GST‐PRSV HC‐Pro‐bound proteins (lane 5) were then analysed by 10% sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and probed with streptavidin‐alkaline phosphatase (streptavidin‐AP) (A) or anti‐GST antibody (B) (GST, 26.0 kDa; GST‐PRSV HC‐Pro, 78.0 kDa; biotinylated PaCRT, 48.2 kDa). The purified GST (lane 1), purified GST‐PRSV HC‐Pro (lane 2) and in vitro translated biotinylated PaCRT (lane 3) were used as controls, respectively. The prestained dual colour protein size marker (Tiangen) is indicated on the left of the panel.

    Article Snippet: The biotinylated proteins can be visualized by binding streptavidin–AP, followed by colorimetric detection, according to the protocol for the Transcend nonradioactive translation detection system (Promega).

    Techniques: In Vitro, Protein Binding, Incubation, Purification, Polyacrylamide Gel Electrophoresis, SDS Page, Marker

    Flow cytometry competition analysis with increasing concentration of the unlabeled (A) or biotinylated (B) foldback diabody anti‐human CCR4 immunotoxins to block the binding of the saturating concentration of the two anti‐human CCR4 mAbs (clone#205410 and L291H4) to the monkey CCR4+ PBMC. Monkey PBMC was incubated with the unlabeled (A) or biotinylated (B) foldback diabody anti‐human immunotoxin at 4 °C for 1 h and washed twice, then stained with the anti‐human CCR4 mAb (A) or the anti‐human CCR4 mAb and APC‐streptavidin (B) at 4 °C for 30 min. The data are representative of two individual experiments.

    Journal: Molecular Oncology

    Article Title: Treg depletion in non-human primates using a novel diphtheria toxin-based anti-human CCR4 immunotoxin

    doi: 10.1016/j.molonc.2015.11.008

    Figure Lengend Snippet: Flow cytometry competition analysis with increasing concentration of the unlabeled (A) or biotinylated (B) foldback diabody anti‐human CCR4 immunotoxins to block the binding of the saturating concentration of the two anti‐human CCR4 mAbs (clone#205410 and L291H4) to the monkey CCR4+ PBMC. Monkey PBMC was incubated with the unlabeled (A) or biotinylated (B) foldback diabody anti‐human immunotoxin at 4 °C for 1 h and washed twice, then stained with the anti‐human CCR4 mAb (A) or the anti‐human CCR4 mAb and APC‐streptavidin (B) at 4 °C for 30 min. The data are representative of two individual experiments.

    Article Snippet: APC‐anti‐human CD8 mAb (clone# RPA‐T8, cat# 301014), PE‐CD20 (clone# 2H7, cat#302306), Biotin‐anti‐human CD16 mAb (clone# 3G8, cat# 302004), PE‐CD16 mAb (clone# 3G8, cat# 302008), FITC‐anti‐human CD14 mAb (clone# M5E2, cat# 301803), PerCp‐Cy5.5‐anti‐human CD11b (clone# M1/70, cat# 101228), PE‐anti‐human CD194 (CCR4) mAb (Clone# L291H4, cat# 359412), PE‐mouse IgG1, κ (clone# MOPC‐21, cat# 400139), Alexa Fluor® 647 anti‐human Foxp3 mAb (clone# 150D, cat# 320014), Alex Fluor 647 Mouse IgG1 κ (clone# MOPC‐21, cat# 400136), PE‐streptavidin (cat# 405204) and APC‐streptavidin (cat# 405207) were purchased from Biolegend.

    Techniques: Flow Cytometry, Cytometry, Concentration Assay, Blocking Assay, Binding Assay, Incubation, Staining

    In vitro binding and depletion analysis of the anti‐human CCR4 immunotoxins to monkey CCR4 + PBMC. (A) Flow cytometry binding analysis of the anti‐human CCR4 immunotoxins to CCR4 + ) was included as a negative control for background due to protein biotinylation. (B) In vitro depletion of the CCR4 + cells within monkey PBMC using the anti‐human CCR4 immunotoxins. Monkey PBMC was incubated with the unlabeled anti‐human CCR4 immunotoxin at 37 °C for 48 h and analyzed by flow cytometry using PE anti‐human CCR4 mAb (clone# L291H4). First panel: monovalent anti‐human CCR4 immunotoxin [DT390‐scFv (1567)]; second panel: bivalent anti‐human CCR4 immunotoxin [DT390‐BiscFv (1567)]; third panel: single‐chain foldback diabody anti‐human CCR4 immunotoxin. PE anti‐human CCR4 antibody (clone #L291H4) for the CCR4 positive control, PE‐mouse IgG1, κ as isotype control of PE‐anti‐human CCR4 mAb (clone# L291H4), APC anti‐human CD4 mAb (clone# L200) for the CD4 positive control, APC mouse IgG1 κ (clone# MOPC‐21) for the isotype control of APC anti‐human CD4 mAb (clone# L200). DT390 (100 nM) was included as negative depleter control. Propidium iodide was added before running the FACS machine for gating the living cells. All control cells in Figure 2B were also incubated at 37 °C for 48 h. (C) Flow cytometry binding analysis of the anti‐human CCR4 immunotoxins to the Foxp3 + CCR4 + monkey PBMC. Monkey PBMC was stained with Alexa Fluor © 647 anti‐human Foxp3 mAb (clone# 150D) and biotinylated anti‐human CCR4 immunotoxin. First panel: monovalent anti‐human CCR4 immunotoxin [DT390‐scFv(1567)]; second panel: bivalent anti‐human CCR4 immunotoxin [DT390‐BiscFv(1567)]; third panel: single‐chain foldback diabody anti‐human CCR4 immunotoxin. Monkey PBMC with only the secondary staining (PE‐conjugated streptavidin) served as the negative control, human CCR4 fluorescein mAb (clone#205410) as the CCR4 positive control, mouse IgG2B fluorescein for the isotype control of human CCR4 fluorescein mAb (clone#205410), Alexa Fluor © 647 anti‐human Foxp3 mAb (clone# 150D) for the Foxp3 positive control, Alex Fluor 647 Mouse IgG1 κ (clone# MOPC‐21) for the isotype control of Alexa Fluor © ) was included as a negative control for background due to protein biotinylation. All Figure 2C analysis was CD4 gated. All of the data (A‐C) are representative of three individual experiments.

    Journal: Molecular Oncology

    Article Title: Treg depletion in non-human primates using a novel diphtheria toxin-based anti-human CCR4 immunotoxin

    doi: 10.1016/j.molonc.2015.11.008

    Figure Lengend Snippet: In vitro binding and depletion analysis of the anti‐human CCR4 immunotoxins to monkey CCR4 + PBMC. (A) Flow cytometry binding analysis of the anti‐human CCR4 immunotoxins to CCR4 + ) was included as a negative control for background due to protein biotinylation. (B) In vitro depletion of the CCR4 + cells within monkey PBMC using the anti‐human CCR4 immunotoxins. Monkey PBMC was incubated with the unlabeled anti‐human CCR4 immunotoxin at 37 °C for 48 h and analyzed by flow cytometry using PE anti‐human CCR4 mAb (clone# L291H4). First panel: monovalent anti‐human CCR4 immunotoxin [DT390‐scFv (1567)]; second panel: bivalent anti‐human CCR4 immunotoxin [DT390‐BiscFv (1567)]; third panel: single‐chain foldback diabody anti‐human CCR4 immunotoxin. PE anti‐human CCR4 antibody (clone #L291H4) for the CCR4 positive control, PE‐mouse IgG1, κ as isotype control of PE‐anti‐human CCR4 mAb (clone# L291H4), APC anti‐human CD4 mAb (clone# L200) for the CD4 positive control, APC mouse IgG1 κ (clone# MOPC‐21) for the isotype control of APC anti‐human CD4 mAb (clone# L200). DT390 (100 nM) was included as negative depleter control. Propidium iodide was added before running the FACS machine for gating the living cells. All control cells in Figure 2B were also incubated at 37 °C for 48 h. (C) Flow cytometry binding analysis of the anti‐human CCR4 immunotoxins to the Foxp3 + CCR4 + monkey PBMC. Monkey PBMC was stained with Alexa Fluor © 647 anti‐human Foxp3 mAb (clone# 150D) and biotinylated anti‐human CCR4 immunotoxin. First panel: monovalent anti‐human CCR4 immunotoxin [DT390‐scFv(1567)]; second panel: bivalent anti‐human CCR4 immunotoxin [DT390‐BiscFv(1567)]; third panel: single‐chain foldback diabody anti‐human CCR4 immunotoxin. Monkey PBMC with only the secondary staining (PE‐conjugated streptavidin) served as the negative control, human CCR4 fluorescein mAb (clone#205410) as the CCR4 positive control, mouse IgG2B fluorescein for the isotype control of human CCR4 fluorescein mAb (clone#205410), Alexa Fluor © 647 anti‐human Foxp3 mAb (clone# 150D) for the Foxp3 positive control, Alex Fluor 647 Mouse IgG1 κ (clone# MOPC‐21) for the isotype control of Alexa Fluor © ) was included as a negative control for background due to protein biotinylation. All Figure 2C analysis was CD4 gated. All of the data (A‐C) are representative of three individual experiments.

    Article Snippet: APC‐anti‐human CD8 mAb (clone# RPA‐T8, cat# 301014), PE‐CD20 (clone# 2H7, cat#302306), Biotin‐anti‐human CD16 mAb (clone# 3G8, cat# 302004), PE‐CD16 mAb (clone# 3G8, cat# 302008), FITC‐anti‐human CD14 mAb (clone# M5E2, cat# 301803), PerCp‐Cy5.5‐anti‐human CD11b (clone# M1/70, cat# 101228), PE‐anti‐human CD194 (CCR4) mAb (Clone# L291H4, cat# 359412), PE‐mouse IgG1, κ (clone# MOPC‐21, cat# 400139), Alexa Fluor® 647 anti‐human Foxp3 mAb (clone# 150D, cat# 320014), Alex Fluor 647 Mouse IgG1 κ (clone# MOPC‐21, cat# 400136), PE‐streptavidin (cat# 405204) and APC‐streptavidin (cat# 405207) were purchased from Biolegend.

    Techniques: In Vitro, Binding Assay, Flow Cytometry, Cytometry, Negative Control, Incubation, Positive Control, FACS, Staining

    Defective 4-1BB activity in T cells and DCs from Gal-9 −/− mice. (a) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD8 + T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (b) 4-1BB and OX40 surface expression on WT and Gal-9 −/− CD8 + T cells activated as in panel a. Mean fluorescent intensity indicated for 4-1BB. Shade, isotype control. (c) Binding of anti–4-1BB (clone 3H3) to activated CD8 + T cells. Activated CD8 + T cells as in panel a were sorted for identical expression of 4-1BB using biotin-anti–4-1BB (Syrian hamster IgG; clone 17B5) and streptavidin-APC. Cells were then stained with unlabeled anti–4-1BB (rat IgG; clone 3H3) and anti–rat IgG FITC to detect anti–4-1BB (clone 3H3) binding to cells. Shade, isotype controls. Data are representative of two different experiments. (d) IFN-γ production by preactivated CD8 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (postsort FACS plot shown) and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of at least three different experiments. (e and f) CD8 + T cells from WT or Gal-9 −/− mice were activated as in panel a, sorted for identical expression of 4-1BB, and recultured for another 24 h to assay IFN-γ production in response to irradiated 4-1BBL + hybridoma cells (e) or varying concentrations of anti-polyhistidine cross-linked r4-1BBL (f). Neutralizing anti–4-1BBL or rat IgG was added to cultures in e. Data are means ± SEM from triplicate cultures and representative of two different experiments. (g, left) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD4 T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (right) IL-2 production by preactivated CD4 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (pre- and postsort FACS plot shown), and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of three different experiments. (h) Surface expression of 4-1BB and Gal-9 (top) and 4-1BB and MHC II (bottom) on ex vivo WT mesenteric LN DCs and splenic DCs from WT and Gal-9 −/− mice cultured with GM-CSF, respectively. Shade, isotype control. (i) ALDEFLUOR staining in preactivated spleen DCs from WT and Gal-9 −/− mice, sorted for identical expression of 4-1BB, and recultured for 24 h in the presence of zymosan with control rat IgG (left) or agonist anti–4-1BB (right). Numbers indicate percentage of cells in each quadrant. Data are representative of three different experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Galectin-9 controls the therapeutic activity of 4-1BB–targeting antibodies

    doi: 10.1084/jem.20132687

    Figure Lengend Snippet: Defective 4-1BB activity in T cells and DCs from Gal-9 −/− mice. (a) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD8 + T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (b) 4-1BB and OX40 surface expression on WT and Gal-9 −/− CD8 + T cells activated as in panel a. Mean fluorescent intensity indicated for 4-1BB. Shade, isotype control. (c) Binding of anti–4-1BB (clone 3H3) to activated CD8 + T cells. Activated CD8 + T cells as in panel a were sorted for identical expression of 4-1BB using biotin-anti–4-1BB (Syrian hamster IgG; clone 17B5) and streptavidin-APC. Cells were then stained with unlabeled anti–4-1BB (rat IgG; clone 3H3) and anti–rat IgG FITC to detect anti–4-1BB (clone 3H3) binding to cells. Shade, isotype controls. Data are representative of two different experiments. (d) IFN-γ production by preactivated CD8 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (postsort FACS plot shown) and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of at least three different experiments. (e and f) CD8 + T cells from WT or Gal-9 −/− mice were activated as in panel a, sorted for identical expression of 4-1BB, and recultured for another 24 h to assay IFN-γ production in response to irradiated 4-1BBL + hybridoma cells (e) or varying concentrations of anti-polyhistidine cross-linked r4-1BBL (f). Neutralizing anti–4-1BBL or rat IgG was added to cultures in e. Data are means ± SEM from triplicate cultures and representative of two different experiments. (g, left) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD4 T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (right) IL-2 production by preactivated CD4 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (pre- and postsort FACS plot shown), and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of three different experiments. (h) Surface expression of 4-1BB and Gal-9 (top) and 4-1BB and MHC II (bottom) on ex vivo WT mesenteric LN DCs and splenic DCs from WT and Gal-9 −/− mice cultured with GM-CSF, respectively. Shade, isotype control. (i) ALDEFLUOR staining in preactivated spleen DCs from WT and Gal-9 −/− mice, sorted for identical expression of 4-1BB, and recultured for 24 h in the presence of zymosan with control rat IgG (left) or agonist anti–4-1BB (right). Numbers indicate percentage of cells in each quadrant. Data are representative of three different experiments.

    Article Snippet: The following antibodies were used for flow cytometry: PE-conjugated anti–Gal-9 (108A2) and anti-CD25 (PC61), PE-Cy7–conjugated anti-NK1.1 (PK136) and anti–IFN-γ (XMG1.2), FITC-conjugated anti-CD44 and anti-CD49b (DX5), peridinin chlorophyll protein–conjugated CD62L (MEL-14), peridinin chlorophyll protein–Cy5.5-conjugated CD8β and anti–L-17A, Pacific blue–conjugated anti-CD3ε (145-2C11) and anti-CD4 (GK1.5), biotin-conjugated anti–4-1BB and anti-OX40, allophycocyanin-conjugated anti–Gr-1 (RB6-8C5), anti–rat IgG (Poly4054), and streptavidin (all from BioLegend); PE-conjugated anti–4-1BBL (TKS-1) and anti–Gal-3 (M3/38), Alexa Flour 700–conjugated MHC class II (M5/114.15.2), and FITC-conjugated anti-CD11c (N418; all from eBioscience).

    Techniques: Activity Assay, Mouse Assay, Expressing, In Vitro, Binding Assay, Staining, FACS, Irradiation, Ex Vivo, Cell Culture

    Generation of cTag8-Expressing 293T Cells for Modified Vector Production (A) Both 293T (non-transduced) and 293T cells expressing cTag8 (cTag8 293T) by γ-retroviral transduction with cTag8 co-expressed with EGFP were stained with streptavidin-APC for cTag8 expression analysis by flow cytometry. (B) Surface expression analysis of cTag8 by immunofluorescence staining of cTag8 293T cells with streptavidin-APC. Engineered cells were assessed for LV packaging capacity by the production of LVs from both 293T cells (non-modified, NM LVs) and cTag8 293T cells (cTag8 LVs) in plain DMEM, pseudotyped with either RDpro, MLV-ampho, or VSV-G glycoproteins. (C) Viral supernatants were frozen, viral titers (infectious units (IU)/mL) of stocks were determined by infectivity assay, and mean values are presented ± SD of triplicate determinations. (D) Sucrose-cushion-ultracentrifuge-purified VSV-G pseudotyped NM LVs and cTag8 LVs were negatively stained and analyzed by transmission electron microscopy (TEM). Scale bars, 200 nm.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Lentiviral Vector Purification Using Genetically Encoded Biotin Mimic in Packaging Cell

    doi: 10.1016/j.omtm.2018.10.008

    Figure Lengend Snippet: Generation of cTag8-Expressing 293T Cells for Modified Vector Production (A) Both 293T (non-transduced) and 293T cells expressing cTag8 (cTag8 293T) by γ-retroviral transduction with cTag8 co-expressed with EGFP were stained with streptavidin-APC for cTag8 expression analysis by flow cytometry. (B) Surface expression analysis of cTag8 by immunofluorescence staining of cTag8 293T cells with streptavidin-APC. Engineered cells were assessed for LV packaging capacity by the production of LVs from both 293T cells (non-modified, NM LVs) and cTag8 293T cells (cTag8 LVs) in plain DMEM, pseudotyped with either RDpro, MLV-ampho, or VSV-G glycoproteins. (C) Viral supernatants were frozen, viral titers (infectious units (IU)/mL) of stocks were determined by infectivity assay, and mean values are presented ± SD of triplicate determinations. (D) Sucrose-cushion-ultracentrifuge-purified VSV-G pseudotyped NM LVs and cTag8 LVs were negatively stained and analyzed by transmission electron microscopy (TEM). Scale bars, 200 nm.

    Article Snippet: To block non-specific binding, PBS + 0.1% BSA was added for 30 min. Streptavidin-APC (BioLegend) was then added in blocking solution at 1:100 for 1 hr.

    Techniques: Expressing, Modification, Plasmid Preparation, Transduction, Staining, Flow Cytometry, Cytometry, Immunofluorescence, Infection, Purification, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy

    Reversible Streptavidin Binding of Synthetic Surface Expressing Biotin Mimics (A) Schematic diagrams of the chosen mimics with their reported dissociation constants (K D ). Four surface expression structures were cloned, and their architectures are represented: (1) Flush, consisting of a CD8-derived transmembrane (TM) and endodomain (Endo); (2) GPI, consisting of GPI anchor sequence (25 amino acids [aa]), which leads to the addition of GPI at the anchor sequence, with a serine-glycine linker (6 aa); (3) x2-GPI, consisting of 2 copies of the epitopes’ open reading frame (ORF) separated by a serine-glycine linker with the first 14 aa of the CD8α stalk ectodomain (Ecto; Linker_CD8), on a GPI anchor sequence; (4) CD8α, consisting of the CD8α stalk comprising the ecto-, transmembrane, and endodomains of the human CD8α molecule, with a serine-glycine linker. All peptides were cloned into a retroviral plasmid termed “SFG,” derived from the Moloney murine leukemia virus (MoMLV), upstream of the EGFP marker gene expressed by an internal ribosome entry site (IRES). (B) 293T cells were transiently transfected with all cloned constructs and stained with APC-conjugated streptavidin 48 hr later. The median fluorescence intensity (MedFI) of streptavidin binding of EGFP-positive cells are presented in a graph indicating ±SD of three independent transient expression experiments; ****p

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Lentiviral Vector Purification Using Genetically Encoded Biotin Mimic in Packaging Cell

    doi: 10.1016/j.omtm.2018.10.008

    Figure Lengend Snippet: Reversible Streptavidin Binding of Synthetic Surface Expressing Biotin Mimics (A) Schematic diagrams of the chosen mimics with their reported dissociation constants (K D ). Four surface expression structures were cloned, and their architectures are represented: (1) Flush, consisting of a CD8-derived transmembrane (TM) and endodomain (Endo); (2) GPI, consisting of GPI anchor sequence (25 amino acids [aa]), which leads to the addition of GPI at the anchor sequence, with a serine-glycine linker (6 aa); (3) x2-GPI, consisting of 2 copies of the epitopes’ open reading frame (ORF) separated by a serine-glycine linker with the first 14 aa of the CD8α stalk ectodomain (Ecto; Linker_CD8), on a GPI anchor sequence; (4) CD8α, consisting of the CD8α stalk comprising the ecto-, transmembrane, and endodomains of the human CD8α molecule, with a serine-glycine linker. All peptides were cloned into a retroviral plasmid termed “SFG,” derived from the Moloney murine leukemia virus (MoMLV), upstream of the EGFP marker gene expressed by an internal ribosome entry site (IRES). (B) 293T cells were transiently transfected with all cloned constructs and stained with APC-conjugated streptavidin 48 hr later. The median fluorescence intensity (MedFI) of streptavidin binding of EGFP-positive cells are presented in a graph indicating ±SD of three independent transient expression experiments; ****p

    Article Snippet: To block non-specific binding, PBS + 0.1% BSA was added for 30 min. Streptavidin-APC (BioLegend) was then added in blocking solution at 1:100 for 1 hr.

    Techniques: Binding Assay, Expressing, Clone Assay, Derivative Assay, Sequencing, Plasmid Preparation, Marker, Transfection, Construct, Staining, Fluorescence

    SipA binds polymyxin B and LL-37 in vitro and in vivo . Wells from a 96-well plate were coated with either purified SipA or VC1638 (A,B) . LL-37 (A) or biotin-labeled polymyxin B (B) were added to plates and incubated for 1 h. Levels of binding were detected via (A) anti-LL-37 antibody followed by IgG-HRP or (B) Streptavidin-HRP. VC1638 served as a control protein. (C) Coimmunoprecipitation of LL-37 in the presence and absence of 6×His- sipA . Each culture was crosslinked and lysed before loading onto a Ni-NTA column (input), followed by elution with imidazole (output). Blots were probed with anti-LL-37 antibody. Densitometry of the LL-37 band was calculated based on three separate experiments and normalized to the strain containing empty vector. ∗ p =

    Journal: Frontiers in Microbiology

    Article Title: A Periplasmic Antimicrobial Peptide-Binding Protein Is Required for Stress Survival in Vibrio cholerae

    doi: 10.3389/fmicb.2019.00161

    Figure Lengend Snippet: SipA binds polymyxin B and LL-37 in vitro and in vivo . Wells from a 96-well plate were coated with either purified SipA or VC1638 (A,B) . LL-37 (A) or biotin-labeled polymyxin B (B) were added to plates and incubated for 1 h. Levels of binding were detected via (A) anti-LL-37 antibody followed by IgG-HRP or (B) Streptavidin-HRP. VC1638 served as a control protein. (C) Coimmunoprecipitation of LL-37 in the presence and absence of 6×His- sipA . Each culture was crosslinked and lysed before loading onto a Ni-NTA column (input), followed by elution with imidazole (output). Blots were probed with anti-LL-37 antibody. Densitometry of the LL-37 band was calculated based on three separate experiments and normalized to the strain containing empty vector. ∗ p =

    Article Snippet: Plates were washed and either LL-37 or biotinylated polymyxin B were added at 10 μg/mL and incubated for 1 h. Plates were washed again and anti-LL-37 (mouse; Santa-Cruz Biotechnology), was added for 1 h. After washing, either Goat pAb to Mouse IgG + IgM HRP (Abcam), or Strepavidin-HRP (Abcam) was added.

    Techniques: In Vitro, In Vivo, Purification, Labeling, Incubation, Binding Assay, Plasmid Preparation

    Imetelstat (GRN163L) is a competitive inhibitor of primer-substrate binding by telomerase. (A) Experimental design of single-molecule telomerase primer binding and activity assay. Halo-telomerase is modified with a biotin-HaloTag-ligand and immobilized on the coverslip surface using NeutrAvidin. Primer binding is visualized by telomerase-dependent recruitment of a fluorescent primer to the coverslip surface. The telomerase extension product is detected using a fluorescently labeled oligonucleotide anti-sense to the telomerase extension product. (B) Western blot and fluorescence imaging of Halo-telomerase modified with a fluorescent dye (JF646) or biotin, probed with an anti-TERT antibody or HRP-conjugated streptavidin. (C) Single-molecule TIRF imaging of primer molecules recruited to the coverslip surface by telomerase (top) and its colocalization with telomerase extension products after incubation with nucleotide substrate (bottom). (D) Single-molecule TIRF imaging of primer binding by telomerase in the presence of increasing concentrations of imetelstat. (E) Quantification of primer binding to telomerase as a function of imetelstat concentration ( n = 5 fields of view per concentration, data points plotted as mean ± SD, error on IC 50 reflects error in the corresponding fit of the data to a simple binding curve). (F) Direct telomerase assay at 150 mM KCl in the absence and presence of imetelstat (10 nM), or mismatched control oligonucleotide (MM Control, 10 nM), and increasing concentrations of primer substrate. LC1, LC2, and LC3, labeled DNA loading controls. (G) Quantification of telomerase activity as a function of primer concentration in absence and presence of imetelstat (10 nM) or mismatched control oligonucleotide (MM Control, 10 nM).

    Journal: Molecular Biology of the Cell

    Article Title: Dynamics of human telomerase recruitment depend on template-telomere base pairing

    doi: 10.1091/mbc.E17-11-0637

    Figure Lengend Snippet: Imetelstat (GRN163L) is a competitive inhibitor of primer-substrate binding by telomerase. (A) Experimental design of single-molecule telomerase primer binding and activity assay. Halo-telomerase is modified with a biotin-HaloTag-ligand and immobilized on the coverslip surface using NeutrAvidin. Primer binding is visualized by telomerase-dependent recruitment of a fluorescent primer to the coverslip surface. The telomerase extension product is detected using a fluorescently labeled oligonucleotide anti-sense to the telomerase extension product. (B) Western blot and fluorescence imaging of Halo-telomerase modified with a fluorescent dye (JF646) or biotin, probed with an anti-TERT antibody or HRP-conjugated streptavidin. (C) Single-molecule TIRF imaging of primer molecules recruited to the coverslip surface by telomerase (top) and its colocalization with telomerase extension products after incubation with nucleotide substrate (bottom). (D) Single-molecule TIRF imaging of primer binding by telomerase in the presence of increasing concentrations of imetelstat. (E) Quantification of primer binding to telomerase as a function of imetelstat concentration ( n = 5 fields of view per concentration, data points plotted as mean ± SD, error on IC 50 reflects error in the corresponding fit of the data to a simple binding curve). (F) Direct telomerase assay at 150 mM KCl in the absence and presence of imetelstat (10 nM), or mismatched control oligonucleotide (MM Control, 10 nM), and increasing concentrations of primer substrate. LC1, LC2, and LC3, labeled DNA loading controls. (G) Quantification of telomerase activity as a function of primer concentration in absence and presence of imetelstat (10 nM) or mismatched control oligonucleotide (MM Control, 10 nM).

    Article Snippet: The HaloTag modified with biotin was detected using Strepavidin-HRP (Pierce; 1:2000).

    Techniques: Binding Assay, Activity Assay, Modification, Labeling, Western Blot, Fluorescence, Imaging, Incubation, Concentration Assay, Telomerase Assay

    Effect of two SpA derivatives on the binding to coated hIgMs and on the T cell presentation of toxin α. (A) Mα2-3 was incubated overnight in the presence or absence of fixed concentrations of toxin α biotinylated at the NH 2 terminus (Alphabiot) and either ZZ or BB in BSA-coated microwell plates. The solutions were transferred in hIgM-coated plates. Binding of Mα2-3 to the wells was determined using a goat anti–mouse IgG peroxidase conjugate (GAM–PO), whereas binding of biotinylated toxin α was determined using a streptavidin peroxidase conjugate (SA–PO). (B) Toxin α (alpha) was serially diluted and incubated overnight at 4°C in the presence or absence of mAb Mα2-3 (25 nM final) and either ZZ or BB (0.1 μM final for each derivative). 5 × 10 5 splenocytes from BALB/c mice were then added to each well in the presence of 5 × 10 4 T1B2. T cell stimulation was assessed as previously described.

    Journal: The Journal of Experimental Medicine

    Article Title: Presentation of Antigen in Immune Complexes Is Boosted by Soluble Bacterial Immunoglobulin Binding Proteins

    doi:

    Figure Lengend Snippet: Effect of two SpA derivatives on the binding to coated hIgMs and on the T cell presentation of toxin α. (A) Mα2-3 was incubated overnight in the presence or absence of fixed concentrations of toxin α biotinylated at the NH 2 terminus (Alphabiot) and either ZZ or BB in BSA-coated microwell plates. The solutions were transferred in hIgM-coated plates. Binding of Mα2-3 to the wells was determined using a goat anti–mouse IgG peroxidase conjugate (GAM–PO), whereas binding of biotinylated toxin α was determined using a streptavidin peroxidase conjugate (SA–PO). (B) Toxin α (alpha) was serially diluted and incubated overnight at 4°C in the presence or absence of mAb Mα2-3 (25 nM final) and either ZZ or BB (0.1 μM final for each derivative). 5 × 10 5 splenocytes from BALB/c mice were then added to each well in the presence of 5 × 10 4 T1B2. T cell stimulation was assessed as previously described.

    Article Snippet: Streptavidin-PE (SAPE) was purchased from Caltag Labs.

    Techniques: Binding Assay, Incubation, Mouse Assay, Cell Stimulation

    The concept of a high-throughput screening method to identify compounds or proteins targeting protein–nucleic acids interactions. The first step is to link a biotin-labeled oligomer to surface of a multiple-well plate through biotin–streptavidin interaction. After the surface-blocking step, a nucleic acids–binding protein is added to the wells to bind to the oligomer. The compounds or proteins of interest can also be added to the wells to inhibit or enhance the protein binding, which is the basis of the method for high-throughput drug screening. Colorimetric, chemiluminescence or fluorescence methods can be used to detect whether the compounds or proteins inhibit or enhance the binding capacities of the nucleic acids–binding protein.

    Journal: Nucleic Acids Research

    Article Title: A rapid and sensitive high-throughput screening method to identify compounds targeting protein–nucleic acids interactions

    doi: 10.1093/nar/gkv069

    Figure Lengend Snippet: The concept of a high-throughput screening method to identify compounds or proteins targeting protein–nucleic acids interactions. The first step is to link a biotin-labeled oligomer to surface of a multiple-well plate through biotin–streptavidin interaction. After the surface-blocking step, a nucleic acids–binding protein is added to the wells to bind to the oligomer. The compounds or proteins of interest can also be added to the wells to inhibit or enhance the protein binding, which is the basis of the method for high-throughput drug screening. Colorimetric, chemiluminescence or fluorescence methods can be used to detect whether the compounds or proteins inhibit or enhance the binding capacities of the nucleic acids–binding protein.

    Article Snippet: Materials Biotin-labeled hairpin DNA oligomer FL814 containing a specific binding site of HMGA2 was purchased from Eurofins MWG Operon, Inc. Streptavidin covalently coated 96-well plates (NUNC Immobilizer Streptavidin-F96 clear) were from Thermo Fisher Scientific, Inc. Antibody against HMGA2 (HMGA2 (D1A7) Rabbit mAb) and Anti-rabbit IgG, HRP-linked Antibody #7074 were purchased from Cell Signaling, Inc. Ultra TMB-ELISA was bought from Thermo Fisher Scientific, Inc.

    Techniques: High Throughput Screening Assay, Labeling, Blocking Assay, Binding Assay, Protein Binding, Fluorescence

    A MACS selection procedure for the prospective isolation of progenitor cells for beige/brite differentiation . (A–F) Single cell suspensions were obtained by collagenase digestion of subcutaneous fat, and stained for six-color FACS with the indicated antibodies after removal of adipocytes by centrifugation and erythrocytes by TER119-MACS ® depletion. Debris and singlets were excluded and selected through FSC/SSC and FSC-A/H, respectively. (A–C) and (D–F) represent independent gating schemes. Values (%) indicate cells % of parent plot. Representative plots from multiple independent experiments are shown. (G) Erythrocytes, leukocytes, and endothelial cells were removed from single cell suspensions in a first MACS step with biotinylated Ter119, CD45, and CD31 antibodies and streptavidin-conjugated microbeads. Sca-1 + cells were enriched in a second MACS step with Sca-1-PE-Cy7 antibody and anti-PE-Cy7 microbeads. The resulting cell population (Lin − Sca-1 + eluate) as well as the Lin − flow-through were subjected to flow cytometry. Comparable purities were obtained with directly conjugated antibody-bead combinations (data not shown). (H) MACS- and FACS-purified cells were cultured and differentiated for 8 days in the presence or absence of cPGI 2 (see Materials and Methods ) before subjected to RNA expression analysis by qRT-PCR (* indicates t -test p

    Journal: Frontiers in Endocrinology

    Article Title: Transcriptional Pathways in cPGI2-Induced Adipocyte Progenitor Activation for Browning

    doi: 10.3389/fendo.2015.00129

    Figure Lengend Snippet: A MACS selection procedure for the prospective isolation of progenitor cells for beige/brite differentiation . (A–F) Single cell suspensions were obtained by collagenase digestion of subcutaneous fat, and stained for six-color FACS with the indicated antibodies after removal of adipocytes by centrifugation and erythrocytes by TER119-MACS ® depletion. Debris and singlets were excluded and selected through FSC/SSC and FSC-A/H, respectively. (A–C) and (D–F) represent independent gating schemes. Values (%) indicate cells % of parent plot. Representative plots from multiple independent experiments are shown. (G) Erythrocytes, leukocytes, and endothelial cells were removed from single cell suspensions in a first MACS step with biotinylated Ter119, CD45, and CD31 antibodies and streptavidin-conjugated microbeads. Sca-1 + cells were enriched in a second MACS step with Sca-1-PE-Cy7 antibody and anti-PE-Cy7 microbeads. The resulting cell population (Lin − Sca-1 + eluate) as well as the Lin − flow-through were subjected to flow cytometry. Comparable purities were obtained with directly conjugated antibody-bead combinations (data not shown). (H) MACS- and FACS-purified cells were cultured and differentiated for 8 days in the presence or absence of cPGI 2 (see Materials and Methods ) before subjected to RNA expression analysis by qRT-PCR (* indicates t -test p

    Article Snippet: The flow-through was collected and stained with CD45-FITC (30-F11, eBioscience), CD31-eFluor 450 (390, eBioscience), CD29-PerCP-eFluor 710 (HMb1-1, eBioscience), CD34-Alexa Fluor 647 (RAM34, BD Biosciences, Heidelberg, Germany), Sca-1-Alexa Fluor 700 (D7, eBioscience), and CD140a(Pdgfrα)-biotin (APA5, eBioscience) for 30 min on ice, followed by staining with streptavidin-PE-Cy7 (eBioscience).

    Techniques: Magnetic Cell Separation, Selection, Isolation, Staining, FACS, Centrifugation, Flow Cytometry, Cytometry, Purification, Cell Culture, RNA Expression, Quantitative RT-PCR