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  • 98
    Vector Laboratories concanavalin a
    CADA specifically inhibits the biogenesis of human CD4. (A, B) CADA inhibits the biosynthesis of CD4. CD4 + .CHO cells were washed and kept in methionine and cysteine-free medium in the presence or absence of 16 µM CADA for 45 min before exposure to [ 35 S]methionine/cysteine (Met/Cys) for 30 min. Pulsed-labelled cells were then washed, lysed, and analyzed directly (A) or incubated in normal medium for up to 4 h (chase) in the presence or absence of 16 µM CADA (B). At specified time points cell lysates were immunoprecipitated for CD4. The flow through fraction (FT) of the CD4-immunoprecipitated samples is also presented. Note that the weaker CD4 bands in the control samples at longer chase time points are the result of the high turnover of hCD4 in CHO cells. Molecular mass is in kDa. (C–F) CD4 negative and stably CD4-YFP transfected CHO cells were pretreated with CADA (5 µM) or DMSO for 1 h before starvation in Met/Cys free medium with CADA, DMSO, or 50 µg/ml CHX. Cells were pulsed for 30 min, washed, and incubated in fresh medium without serum for 90 min. After collection of supernatant proteins (Media) cells were first permeabilized with digitonin buffer to obtain the cytosolic cell fraction before lysis in NP-40 buffer to collect the membrane proteins. Membrane fractions were further incubated with <t>Concanavalin</t> A (ConA) agarose beads (Glycosylated). Molecular mass is in kDa. (D) Quantification of 35 S incorporation in (C) by scintillation counting ( n = 4). NS, not significant; * p
    Concanavalin A, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 98/100, based on 390 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher concanavalin a
    Three-dimensional projections of individual components in biofilms formed by four methicillin-resistant  Staphylococcus aureus  (MRSA) strains on polystyrene after 24 h of incubation. The images were obtained from confocal stack images by the IMARIS 9.1 software, virtual projections being included on the right.  1 propidium iodide;  2 fluorescein isothiocyanate isomer I;  3 DiIC18(5) oil, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate;  4 concanavalin A, tetramethylrhodamine conjugate;  5 calcofluor white M2R.
    Concanavalin A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1028 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore concanavalin a
    4-dpf cells have multiple protrusions because the protrusions are intrinsically small.  (a) Example force–time curve showing a membrane tether force quantification. A concanavalin A–coated AFM cantilever is brought into contact with the cell for 10 s and then withdrawn. At this time, a membrane tether connecting the cell to the cantilever produces a negative force reading. After tether breakage, the force experienced by the cantilever returns to zero. The difference between the prebreakage and postbreakage force is quantified as the tether force. (b) Tether force was measured in 2-dpf ( n  = 25) and 4-dpf single-front ( n  = 8), and 4-dpf multiple-front ( n  = 15) cells. n.s., P  >  0.05 as measured by a two-sample  t  test. (c) Mean protrusion width measured in individual 2-dpf ( n  = 61) and 4-dpf ( n  = 157) multiple-front cells. **, P
    Concanavalin A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 14596 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GE Healthcare con a sepharose
    Effects of 4 M urea on <t>Con</t> A <t>Sepharose.</t> A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.
    Con A Sepharose, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Vector Laboratories biotinylated
    Specificity of HAM/TSP IgG for CNS neurons and isolation of the neuronal antigen. a , Immunohistochemistry of human tissues. <t>Biotinylated</t> HAM/TSP IgG stained CNS neurons (cortex, brown reaction product) (scale bar, 100 µm). There was no staining of glia (arrowhead), dorsal root ganglion or systemic organs. A tax mAb mimicked HAM/TSP IgG staining indicated by the red stain. Neurofilament (gray) confirmed localization of tax mAb to neurons. b , Western blots of proteins derived from CNS neurons. HAM/TSP IgG showed an intense signal at 33 kD (lane 1). There was no reactivity using IgG isolated from HTLV-1 seronegative (lane 2) or HTLV-1 seropositive, neurologically asymptomatic individuals (lane 3). The tax mAb recognized the 33-kD neuronal antigen (lane 4). c , Purification and western blots of the neuronal protein following high salt extraction and centrifugation. Molecular weight markers (lane 1), precipitate (lane 2) and supernatant (lane 3). HAM/TSP IgG reacted with the supernatant (lane 3), but not the precipitate (lane 2). d , 2-D gel electrophoresis and western blot of the neuronal protein purified from the supernatant. Coomassie stain of gel (left). Western blot of the neuronal extract (right), HAM/TSP IgG reacted at 33–38 kD, pI = 9.3 (arrow).
    Biotinylated, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 750 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare concanavalin a
    Down-modulation of B7-molecules and suppression of T-cell proliferation is CTLA-4-dependent. (a) BM-derived DCs (0·5 × 10 6 /ml) were cultured either in medium alone, or with wild type CD4 +  CD25 +  T cells (2 × 10 6 /ml) (CD25 + ), CD4 +  CD25 −  T cells (CD25 − ), or a 1 : 1 mixture of T cells (CD25 +/− ) (2 × 10 6 /ml) and Con A (2·5 µg/ml). Where indicated, anti-CTLA-4 (100 µg/ml) together with Fc-receptor blocking antibody 2.4G2 (2·5 µg/ml) were included in the cultures. The cultures were harvested and the DCs analysed for cell surface expression of CD80 and CD86 after 48 hr of culture. The results are presented as percentage of the GeoMFI (CD80 = 534 and CD86 = 581) obtained from analysis of DCs grown in the absence of T cells. (b) Parallel cultures to those in (a) were assayed for proliferation after 48 hr of culture.  Ctla-4 -deficient CD4 +  CD25 +  T cells are anergic but do not down-modulate B7-molecules on DCs. (c) CD4 +  CD25 +  T cells (2·5 × 10 4 /well) from  Ctla-4 -deficient and normal (wild type) mice were cultured with DCs (1 × 10 4 /well) and anti-CD3 (1 µg/ml) or anti-CD3 + anti-CD28 (5 µg/ml) and proliferation assayed after 72 hr (d) DCs (0·5 × 10 6 /ml) were cultured for 48 hr either in medium alone or with anti-CD3 stimulated (1 µg/ml) CD4 +  CD25 +  T cells (2 × 10 6 /ml) either from wild type,  Cd28 -deficient or  Ctla-4 -deficient mice. Cells were harvested, stained and CD11c +  cells were analysed for CD80 and CD86 cell surface expression by flow cytometry. The results are presented as percentage of the GeoMFI (CD80 = 402 and CD86 = 2184) obtained from analysis of DCs grown in the absence of T cells. In (d) only age-matched wild type controls for the  Ctla-4 -deficient mice are shown, because the adult controls for the  Cd28 -deficient mice gave similar results. The data shown are representative of two to four independent experiments.
    Concanavalin A, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 289 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Valiant concanavalin a
    SPR sensorgrams (without background subtraction) of non-specific BSA (a) and specific Con A (b) binding to mannose functionalized chips with different underlying chemistries. C 11  substrate: 11-mercaptoundecanol, (OEG) 3 -C 11  substrate: (11-mercaptoundecyl)
    Concanavalin A, supplied by Valiant, used in various techniques. Bioz Stars score: 93/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher concanavalin a alexa fluor 488 conjugate
    Characterization of C. albicans and S. gordonii single- and dual-species biofilms formed on titanium using CSLM and 3D reconstruction software. Biofilms were grown for 24 h in BMM synthetic saliva in a 48-well plate containing titanium discs, and stained with Concanavalin <t>A-Alexa</t> Fluor ® 488 conjugate, 1 x FilmTracer™ SYPRO ® Ruby Biofilm Matrix Stain, and DAPI. Scale bar corresponds to 50 µm. Included in the figure are the xy , xz , and yz views of the resulting biofilm.
    Concanavalin A Alexa Fluor 488 Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson concanavalin a
    Sec13 H/− Mice Present Specific Immunological Alterations. ( a ) Analysis of immunological cell populations in Sec13 +/+ and Sec13 H/− mice. Cell subsets isolated from spleen and mesenteric lymph nodes (MLN) were analyzed by flow cytometry using CD4+ and CD8+ as phenotypic markers for T lymphocytes, CD19+ for B lymphocytes, and CD11b+CD11c- for macrophages. Results are the mean ± SD (n = 8). Intracellular cytokine production (IL-6, IL-10, and IL-12) was measured by flow cytometry in cultured cells in the presence of ConA and GolgiStop®. ( b – e ) Splenic cells from Sec13 +/+ and Sec13 H/− mice were subjected to flow cytometry to analyze frequencies of specific populations and expression of molecules. Cells were harvested and cultured for 48h in the presence of <t>Con-A.</t> Intracellular cytokine production was measured by flow cytometry in cultured cells in the presence of ConA and GolgiStop®. Cells were also stained with FITC-labeled anti-CD8, PercP-labeled anti-CD3 and PE-labeled anti-IFN-γ. Bar graphs are shown as mean ± SEM. ( b , c ) Cytokine production by splenic cells was measured in the supernatant by sandwich ELISA. Bar graphs are shown as mean ± SEM. ( d ) MHC I and MHC II expression was measured in CD11b+ CD11- cells isolated from mesenteric lymph nodes, spleen, and peritoneum. Graphs are representative of 6 mice/group. ( e ) Cells were stained with FITC-labeled anti-CD4, PercP-labeled anti-CD25, and PE-labeled anti-LAP. CD4+ cells and CD25+ were gated. LAP+ cells were gated within CD4+ CD25+ cells. Plots are representative of the mean of 3 mice/group. All data are representative of three independent experiments. Bar graphs are shown as mean ± SEM. Student T test was applied. *p
    Concanavalin A, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher concanavalin a alexa fluor 594 conjugate
    Cellular localization of FITC-labeled siRNA (green) following magnetofection (MF; A, B) and lipofection (L2000; C, D) procedures in primary fibroblasts imaged by confocal laser scanning microscopy. Both a siRNA-only control in the absence of transfection reagent (A, 2 μg siRNA; C, 1 μg siRNA) and siRNA/transfection reagent complexes (B, D) were applied. Nuclear DNA was counterstained with DAPI (blue). The cell surface marker, ConA <t>Alexa</t> <t>Fluor</t> 594 (orange-red), was used to stain the cell membrane. The last column (“merge”) shows images from the first three columns superimposed, illustrating intracellular uptake of siRNA/transfection reagent complexes (B, D) and absence of cellular uptake of FITC-siRNA alone (A, C). Co-localization of FITC-siRNA-only or siRNA/transfection reagent complexes with the cell surface did not occur.
    Concanavalin A Alexa Fluor 594 Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    FUJIFILM concanavalin a
    Effect of oral administration of the  L. pentosus  strain S-PT84 on stress-induced immune compromisation of splenocytes. Splenocytes were collected from mice that were fed an AIN-93 M diet with or without S-PT84 for 1 week and then restrained. The number of splenocytes (A), splenic NK activity against target YAC-1 cells (effector:target ratio = 80:1, 40:1, and 20:1) (B), and IFN-γ (C) and IL-4 (D) production levels in splenocytes cultured with Con A (2.5 µg/ml) for 24 hr were measured as described in MATERIALS AND METHODS. Data are presented as the mean ± SE of five mice. Different letters ( a ,  b  and  c ) indicate significant differences between groups (p
    Concanavalin A, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim concanavalin a
    Effect of oral administration of the  L. pentosus  strain S-PT84 on stress-induced immune compromisation of splenocytes. Splenocytes were collected from mice that were fed an AIN-93 M diet with or without S-PT84 for 1 week and then restrained. The number of splenocytes (A), splenic NK activity against target YAC-1 cells (effector:target ratio = 80:1, 40:1, and 20:1) (B), and IFN-γ (C) and IL-4 (D) production levels in splenocytes cultured with Con A (2.5 µg/ml) for 24 hr were measured as described in MATERIALS AND METHODS. Data are presented as the mean ± SE of five mice. Different letters ( a ,  b  and  c ) indicate significant differences between groups (p
    Concanavalin A, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bangs Laboratories concanavalin a coated magnetic beads
    An automated platform for high-throughput  in situ  profiling of chromatin proteins. ( a ) AutoCUT  RUN workflow. (1) Cells or tissue are bound to Concanavalin A-coated beads, permeabilized with digitonin, and incubated with an antibody targeting a chromatin protein. (2) Samples are arrayed in a 96-well plate and (3) processed on a Biomek robot fitted with a 96-well magnetic plate for magnetic separation during washes (α), and an aluminum chiller block (β) routed to a circulating water bath (γ) for temperature control. (4) AutoCUT  RUN produces in 2 days up to 96 libraries that are ready to be pooled and sequenced. ( b ) Hierarchically clustered correlation matrix of AutoCUT  RUN profiles of histone-H3 modifications that mark active (pink) and repressed (blue) chromatin in H1 (orange) and K562 (purple) cells. Pearson correlations were calculated using the log 2  transformed values of read counts split into 500 bp bins across the genome.
    Concanavalin A Coated Magnetic Beads, supplied by Bangs Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore concanavalin a sepharose
    An automated platform for high-throughput  in situ  profiling of chromatin proteins. ( a ) AutoCUT  RUN workflow. (1) Cells or tissue are bound to Concanavalin A-coated beads, permeabilized with digitonin, and incubated with an antibody targeting a chromatin protein. (2) Samples are arrayed in a 96-well plate and (3) processed on a Biomek robot fitted with a 96-well magnetic plate for magnetic separation during washes (α), and an aluminum chiller block (β) routed to a circulating water bath (γ) for temperature control. (4) AutoCUT  RUN produces in 2 days up to 96 libraries that are ready to be pooled and sequenced. ( b ) Hierarchically clustered correlation matrix of AutoCUT  RUN profiles of histone-H3 modifications that mark active (pink) and repressed (blue) chromatin in H1 (orange) and K562 (purple) cells. Pearson correlations were calculated using the log 2  transformed values of read counts split into 500 bp bins across the genome.
    Concanavalin A Sepharose, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Vector Laboratories agarose bound concanavalin a
    Analysis of UT-A1 glycan sialylation in PKC- α  knockout mouse. Kidney IM tissues were dissected from C57B6 wild-type (WT) or PKC- α  knockout mice and processed for lipid raft isolation by 5%–40% sucrose gradient ultracentrifugation. Lipid raft fractions (fractions 2–5) were collected and used for a lectin pull-down assay. UT-A1 proteins from (A) cell membrane lipid rafts and (B) lectin-precipitated samples were examined by Western blot with UT-A1 antibody. Con A, concanavalin A; IB, immunoblot; MAA, maackia amurensis agglutinin; Tomato, lycopersicum esculentum lectin; WGA, wheat germ agglutinin.
    Agarose Bound Concanavalin A, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 94/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher alexa fluor 594 conjugated concanavalin a
    The effect of R164Q mutation on the signal transduction and cell surface expression of PKR2. A , R164Q mutation disrupted the signal transduction of PKR2 as assayed with an aequorin-based assay. B , both WT and R164Q-PKR2 were presented on the cell surface. The <t>Alexa</t> Fluor 594-conjugated <t>concanavalin</t> A was used to label the membrane. The fluorescence was monitored with a confocal microscope. Scale bar: 10 μm.
    Alexa Fluor 594 Conjugated Concanavalin A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher concanavalin a alexa fluor 647 conjugate
    Confocal laser microscopy images of MDA-MB-231 breast cancer cells incubated for 8 hours with PaxOG-eNPs. ( top left ) Intracellular Oregon Green® 488 Taxol signal detected using 488 nm laser. ( top right ) Concanavalin <t>A-Alexa</t> <t>Fluor</t> 647 signal detected using 633 nm laser and marking the plasma membrane in red. ( bottom left ) Differential interference contrast image. ( bottom right ) Composite image showing intracellular delivery of the Oregon Green® 488 Taxol to the cell cytoplasm. Scale bar = 10 μm.
    Concanavalin A Alexa Fluor 647 Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Seikagaku concanavalin a
    Comparison of N-glycan profiles in HepG2 cells treated with and without 2FF. ( A ) MS spectrum of the permethylated glycans from the cells treated with DMSO only (upper) and DMSO containing 2FF (lower). ( B ) Percentages of fucosylated complex glycans in total complex glycans from HepG2 cells. The values were calculated from peak areas in the range of m/z 1,500-3,500 of the MS spectra. ( C ) HepG2 cells were cultured with 2FF for 3 days at different concentrations as indicated, and then equal amounts of cell lysates were probed with Con A, which specifically recognizes alpha-linked mannose and glucose; WGA, which selectively binds to N-Acetyl glucosamine (GlcNAc) and SNA, which specifically recognizes α 2, 6 sialylation. GAPDH was used as a loading control.
    Concanavalin A, supplied by Seikagaku, used in various techniques. Bioz Stars score: 92/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CADA specifically inhibits the biogenesis of human CD4. (A, B) CADA inhibits the biosynthesis of CD4. CD4 + .CHO cells were washed and kept in methionine and cysteine-free medium in the presence or absence of 16 µM CADA for 45 min before exposure to [ 35 S]methionine/cysteine (Met/Cys) for 30 min. Pulsed-labelled cells were then washed, lysed, and analyzed directly (A) or incubated in normal medium for up to 4 h (chase) in the presence or absence of 16 µM CADA (B). At specified time points cell lysates were immunoprecipitated for CD4. The flow through fraction (FT) of the CD4-immunoprecipitated samples is also presented. Note that the weaker CD4 bands in the control samples at longer chase time points are the result of the high turnover of hCD4 in CHO cells. Molecular mass is in kDa. (C–F) CD4 negative and stably CD4-YFP transfected CHO cells were pretreated with CADA (5 µM) or DMSO for 1 h before starvation in Met/Cys free medium with CADA, DMSO, or 50 µg/ml CHX. Cells were pulsed for 30 min, washed, and incubated in fresh medium without serum for 90 min. After collection of supernatant proteins (Media) cells were first permeabilized with digitonin buffer to obtain the cytosolic cell fraction before lysis in NP-40 buffer to collect the membrane proteins. Membrane fractions were further incubated with Concanavalin A (ConA) agarose beads (Glycosylated). Molecular mass is in kDa. (D) Quantification of 35 S incorporation in (C) by scintillation counting ( n = 4). NS, not significant; * p

    Journal: PLoS Biology

    Article Title: Signal Peptide-Binding Drug as a Selective Inhibitor of Co-Translational Protein Translocation

    doi: 10.1371/journal.pbio.1002011

    Figure Lengend Snippet: CADA specifically inhibits the biogenesis of human CD4. (A, B) CADA inhibits the biosynthesis of CD4. CD4 + .CHO cells were washed and kept in methionine and cysteine-free medium in the presence or absence of 16 µM CADA for 45 min before exposure to [ 35 S]methionine/cysteine (Met/Cys) for 30 min. Pulsed-labelled cells were then washed, lysed, and analyzed directly (A) or incubated in normal medium for up to 4 h (chase) in the presence or absence of 16 µM CADA (B). At specified time points cell lysates were immunoprecipitated for CD4. The flow through fraction (FT) of the CD4-immunoprecipitated samples is also presented. Note that the weaker CD4 bands in the control samples at longer chase time points are the result of the high turnover of hCD4 in CHO cells. Molecular mass is in kDa. (C–F) CD4 negative and stably CD4-YFP transfected CHO cells were pretreated with CADA (5 µM) or DMSO for 1 h before starvation in Met/Cys free medium with CADA, DMSO, or 50 µg/ml CHX. Cells were pulsed for 30 min, washed, and incubated in fresh medium without serum for 90 min. After collection of supernatant proteins (Media) cells were first permeabilized with digitonin buffer to obtain the cytosolic cell fraction before lysis in NP-40 buffer to collect the membrane proteins. Membrane fractions were further incubated with Concanavalin A (ConA) agarose beads (Glycosylated). Molecular mass is in kDa. (D) Quantification of 35 S incorporation in (C) by scintillation counting ( n = 4). NS, not significant; * p

    Article Snippet: To isolate glycosylated proteins, total cell lysates or digitonin-resistant membrane fractions were further incubated with Concanavalin A (Vector Laboratories) agarose beads overnight at 4°C by gentle rotation.

    Techniques: Incubation, Immunoprecipitation, Flow Cytometry, Stable Transfection, Transfection, Lysis

    Three-dimensional projections of individual components in biofilms formed by four methicillin-resistant  Staphylococcus aureus  (MRSA) strains on polystyrene after 24 h of incubation. The images were obtained from confocal stack images by the IMARIS 9.1 software, virtual projections being included on the right.  1 propidium iodide;  2 fluorescein isothiocyanate isomer I;  3 DiIC18(5) oil, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate;  4 concanavalin A, tetramethylrhodamine conjugate;  5 calcofluor white M2R.

    Journal: Frontiers in Microbiology

    Article Title: Characterization of Biofilms Formed by Foodborne Methicillin-Resistant Staphylococcus aureus

    doi: 10.3389/fmicb.2018.03004

    Figure Lengend Snippet: Three-dimensional projections of individual components in biofilms formed by four methicillin-resistant Staphylococcus aureus (MRSA) strains on polystyrene after 24 h of incubation. The images were obtained from confocal stack images by the IMARIS 9.1 software, virtual projections being included on the right. 1 propidium iodide; 2 fluorescein isothiocyanate isomer I; 3 DiIC18(5) oil, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate; 4 concanavalin A, tetramethylrhodamine conjugate; 5 calcofluor white M2R.

    Article Snippet: SYTO 9 and propidium iodide (PI) from the LIVE/DEAD® BacLightTM Bacterial Viability Kit, DiIC18(5) oil, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD’oil) and concanavalin A, tetramethylrhodamine conjugate (ConA-TMR) were purchased from Invitrogen (Carlsbad, CA, United States), while fluorescein isothiocyanate isomer I (FITC) and calcofluor white M2R (CFW) were purchased from Sigma (St. Louis, MO, United States).

    Techniques: Incubation, Software

    4-dpf cells have multiple protrusions because the protrusions are intrinsically small.  (a) Example force–time curve showing a membrane tether force quantification. A concanavalin A–coated AFM cantilever is brought into contact with the cell for 10 s and then withdrawn. At this time, a membrane tether connecting the cell to the cantilever produces a negative force reading. After tether breakage, the force experienced by the cantilever returns to zero. The difference between the prebreakage and postbreakage force is quantified as the tether force. (b) Tether force was measured in 2-dpf ( n  = 25) and 4-dpf single-front ( n  = 8), and 4-dpf multiple-front ( n  = 15) cells. n.s., P  >  0.05 as measured by a two-sample  t  test. (c) Mean protrusion width measured in individual 2-dpf ( n  = 61) and 4-dpf ( n  = 157) multiple-front cells. **, P

    Journal: The Journal of Cell Biology

    Article Title: Myosin light chain kinase regulates cell polarization independently of membrane tension or Rho kinase

    doi: 10.1083/jcb.201409001

    Figure Lengend Snippet: 4-dpf cells have multiple protrusions because the protrusions are intrinsically small. (a) Example force–time curve showing a membrane tether force quantification. A concanavalin A–coated AFM cantilever is brought into contact with the cell for 10 s and then withdrawn. At this time, a membrane tether connecting the cell to the cantilever produces a negative force reading. After tether breakage, the force experienced by the cantilever returns to zero. The difference between the prebreakage and postbreakage force is quantified as the tether force. (b) Tether force was measured in 2-dpf ( n = 25) and 4-dpf single-front ( n = 8), and 4-dpf multiple-front ( n = 15) cells. n.s., P > 0.05 as measured by a two-sample t test. (c) Mean protrusion width measured in individual 2-dpf ( n = 61) and 4-dpf ( n = 157) multiple-front cells. **, P

    Article Snippet: Olympus BioLevers (k = 60 pN/nm) were calibrated using the thermal noise method, plasma-cleaned for 30 s, and incubated in 4 mg/ml Concanavalin A (Sigma-Aldrich) for 2 h at 30°C.

    Techniques:

    Interaction between Con A and HuNoV (GII.4). Sensograms obtained by surface plasmon resonance (SPR) analysis using Au sensor chips with a concentration series (250, 125, 62.5, 31.3, 15.6, and 0 nM) of (A) native Con A ( C. ensiformis PDB accession: 3NWK ), (B) metal coordinating region (MCR)-mutated Con A (mCon A MCR ), and (C) carbohydrate binding region (CBR)-mutated Con A (mCon A CBR ) (n = 3, mean values). The interaction model was used to determine the equilibrium dissociation constant (K D ); K D values were 71.5 ± 6.7 nM, 22.0 ± 17.0 μM, and 110.8 ± 7.5 nM for native Con A, mCon A MCR , and mCon A CBR , respectively. Mutations of Con A around the MCR and CBR are shown in (B) and (C).

    Journal: Biomaterials

    Article Title: Exploration of the metal coordination region of concanavalin A for its interaction with human norovirus

    doi: 10.1016/j.biomaterials.2017.03.006

    Figure Lengend Snippet: Interaction between Con A and HuNoV (GII.4). Sensograms obtained by surface plasmon resonance (SPR) analysis using Au sensor chips with a concentration series (250, 125, 62.5, 31.3, 15.6, and 0 nM) of (A) native Con A ( C. ensiformis PDB accession: 3NWK ), (B) metal coordinating region (MCR)-mutated Con A (mCon A MCR ), and (C) carbohydrate binding region (CBR)-mutated Con A (mCon A CBR ) (n = 3, mean values). The interaction model was used to determine the equilibrium dissociation constant (K D ); K D values were 71.5 ± 6.7 nM, 22.0 ± 17.0 μM, and 110.8 ± 7.5 nM for native Con A, mCon A MCR , and mCon A CBR , respectively. Mutations of Con A around the MCR and CBR are shown in (B) and (C).

    Article Snippet: Con A isolated from Canavalia ensiformis (Jack bean, C7275, Sigma, St. Louis, USA) was purchased.

    Techniques: SPR Assay, Concentration Assay, Binding Assay

    ECD mass spectra of intact protein assemblies: (a) concanavalin A (ConA) from Canavalia ensiformis (Jack bean); (b) FMO antenna protein from green sulfur bacterium Chlorobaculum tepidum ; (c) yeast alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae . The insets are native ESI spectra of each protein complex.

    Journal: Analytical chemistry

    Article Title: Native Electrospray and Electron-Capture Dissociation FTICR Mass Spectrometry for Top-down Studies of Protein Assemblies

    doi: 10.1021/ac200695d

    Figure Lengend Snippet: ECD mass spectra of intact protein assemblies: (a) concanavalin A (ConA) from Canavalia ensiformis (Jack bean); (b) FMO antenna protein from green sulfur bacterium Chlorobaculum tepidum ; (c) yeast alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae . The insets are native ESI spectra of each protein complex.

    Article Snippet: Ammonium acetate, water, yeast alcohol dehydrogenase (ADH) form Saccharomyces cerevisiae , concanavalin A (ConA) from Canavalia ensiformis (Jack bean) were purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques:

    β-glucan supplemented diet elicit cytokine production by ConA stimulated lymphocytes. Cytokine production (IL1-β, TNF-α, IL-12, IL-13 and IL-4) in supernatants from Con A treated lymphocytes was quantified with commercial ELISA kits. Absorbance at 450nm was measured and Δ Absorbance data (time 1 minus time 0) were used for comparisons among groups.

    Journal: Food & Nutrition Research

    Article Title: Lentinula edodes β-glucan enriched diet induces pro- and anti-inflammatory macrophages in rabbit

    doi: 10.1080/16546628.2017.1412791

    Figure Lengend Snippet: β-glucan supplemented diet elicit cytokine production by ConA stimulated lymphocytes. Cytokine production (IL1-β, TNF-α, IL-12, IL-13 and IL-4) in supernatants from Con A treated lymphocytes was quantified with commercial ELISA kits. Absorbance at 450nm was measured and Δ Absorbance data (time 1 minus time 0) were used for comparisons among groups.

    Article Snippet: Non-adherent cells containing lymphocytes were seeded at a density of 105 cells/well in 96-well round bottom plates and incubated with 1µg/well of the mitogen Concanavalin A (ConA; Sigma) for 48 hours.

    Techniques: Enzyme-linked Immunosorbent Assay

    Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.

    Journal:

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins

    doi: 10.1016/j.chroma.2007.03.126

    Figure Lengend Snippet: Effects of 4 M urea on Con A Sepharose. A virgin Con A Sepharose column (column filters replaced) was subjected to a standard chromatographic experiment with 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside/4 M urea (pH 4.0) as the eluent.

    Article Snippet: Counterintuitively, higher pyranoside concentrations actually diminished the amount of venom glycoprotein desorbed from Con A Sepharose, regardless of which pyranoside was used ( ).

    Techniques:

    Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been

    Journal:

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins

    doi: 10.1016/j.chroma.2007.03.126

    Figure Lengend Snippet: Influence of pH on venom glycoprotein and PDE desorbed from Con A Sepharose. Eluents consisted of 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside titrated with NaOH to pH 3.0, 4.0, 5.0, or 6.0. Bradford data and PDE activities have been

    Article Snippet: Counterintuitively, higher pyranoside concentrations actually diminished the amount of venom glycoprotein desorbed from Con A Sepharose, regardless of which pyranoside was used ( ).

    Techniques:

    Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene

    Journal:

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins

    doi: 10.1016/j.chroma.2007.03.126

    Figure Lengend Snippet: Influence of auxiliary and alternative eluents on glycoprotein and PDE desorption from Con A Sepharose. Auxiliary eluents were added to the standard 100 mM acetic acid/NaOH/1 M methyl-α- d -glucopyranoside (pH 4.0) buffer. These included 20% ethylene

    Article Snippet: Counterintuitively, higher pyranoside concentrations actually diminished the amount of venom glycoprotein desorbed from Con A Sepharose, regardless of which pyranoside was used ( ).

    Techniques:

    Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay

    Journal:

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins

    doi: 10.1016/j.chroma.2007.03.126

    Figure Lengend Snippet: Influence of eluent NaCl concentration on glycoprotein and PDE desorption. Increasing eluent NaCl concentrations reduces glycoprotein desorption from Con A Sepharose even after the Bradford and PDE assays are corrected for direct effects on the assay

    Article Snippet: Counterintuitively, higher pyranoside concentrations actually diminished the amount of venom glycoprotein desorbed from Con A Sepharose, regardless of which pyranoside was used ( ).

    Techniques: Concentration Assay

    Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M

    Journal:

    Article Title: Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins

    doi: 10.1016/j.chroma.2007.03.126

    Figure Lengend Snippet: Glycoprotein elution profiles with 0-4 pauses introduced during desorption. The 1 mL Con A Sepharose column was equilibrated in 50 mM acetic acid/NaOH/500 mM NaCl/1 mM MnCl 2 /1 mM CaCl 2 /1 mM MgCl 2 (pH 6.0) and desorbed with 100 mM acetic acid/NaOH/1 M

    Article Snippet: Counterintuitively, higher pyranoside concentrations actually diminished the amount of venom glycoprotein desorbed from Con A Sepharose, regardless of which pyranoside was used ( ).

    Techniques:

    EBV BNLF2a blocks peptide transport by TAP. (A) TAP-dependent peptide transport in BNLF2aHA-expressing and control MJS cells was assessed by permeabilizing the cells with streptolysin O and incubating them with a fluoresceinated peptide in the presence or absence of ATP. Translocated peptides that had become glycosylated in the endoplasmic reticulum were recovered by adsorption to concanavalin A–sepharose beads. After elution, the recovered peptide was quantitated by fluorometry in arbitrary units. Error bars represent the SEM of triplicates in a representative experiment. (B) Digitonin lysates of BNLF2aHA- expressing and control MJS-GFP cells were subjected to immunoprecipitation (IP) with antibodies specific for TAP1 (148.3), TAP2 (435.4), tapasin (R.gp46C), HLA class I heavy chains (HC10), and HLA class II DRα chains (DA6-147). Cell lysates and immune complexes were separated by SDS-PAGE, followed by Western blot analysis and staining with anti-HA antibody (12CA5) to detect HA-tagged BNLF2a.

    Journal: The Journal of Experimental Medicine

    Article Title: A CD8+ T cell immune evasion protein specific to Epstein-Barr virus and its close relatives in Old World primates

    doi: 10.1084/jem.20070256

    Figure Lengend Snippet: EBV BNLF2a blocks peptide transport by TAP. (A) TAP-dependent peptide transport in BNLF2aHA-expressing and control MJS cells was assessed by permeabilizing the cells with streptolysin O and incubating them with a fluoresceinated peptide in the presence or absence of ATP. Translocated peptides that had become glycosylated in the endoplasmic reticulum were recovered by adsorption to concanavalin A–sepharose beads. After elution, the recovered peptide was quantitated by fluorometry in arbitrary units. Error bars represent the SEM of triplicates in a representative experiment. (B) Digitonin lysates of BNLF2aHA- expressing and control MJS-GFP cells were subjected to immunoprecipitation (IP) with antibodies specific for TAP1 (148.3), TAP2 (435.4), tapasin (R.gp46C), HLA class I heavy chains (HC10), and HLA class II DRα chains (DA6-147). Cell lysates and immune complexes were separated by SDS-PAGE, followed by Western blot analysis and staining with anti-HA antibody (12CA5) to detect HA-tagged BNLF2a.

    Article Snippet: Peptides that had been glycosylated in the endoplasmic reticulum were isolated from lysates by incubation with concanavalin A–sepharose beads (GE Healthcare).

    Techniques: Expressing, Adsorption, Immunoprecipitation, SDS Page, Western Blot, Staining

    Specificity of HAM/TSP IgG for CNS neurons and isolation of the neuronal antigen. a , Immunohistochemistry of human tissues. Biotinylated HAM/TSP IgG stained CNS neurons (cortex, brown reaction product) (scale bar, 100 µm). There was no staining of glia (arrowhead), dorsal root ganglion or systemic organs. A tax mAb mimicked HAM/TSP IgG staining indicated by the red stain. Neurofilament (gray) confirmed localization of tax mAb to neurons. b , Western blots of proteins derived from CNS neurons. HAM/TSP IgG showed an intense signal at 33 kD (lane 1). There was no reactivity using IgG isolated from HTLV-1 seronegative (lane 2) or HTLV-1 seropositive, neurologically asymptomatic individuals (lane 3). The tax mAb recognized the 33-kD neuronal antigen (lane 4). c , Purification and western blots of the neuronal protein following high salt extraction and centrifugation. Molecular weight markers (lane 1), precipitate (lane 2) and supernatant (lane 3). HAM/TSP IgG reacted with the supernatant (lane 3), but not the precipitate (lane 2). d , 2-D gel electrophoresis and western blot of the neuronal protein purified from the supernatant. Coomassie stain of gel (left). Western blot of the neuronal extract (right), HAM/TSP IgG reacted at 33–38 kD, pI = 9.3 (arrow).

    Journal: Nature medicine

    Article Title: Autoimmunity due to molecular mimicry as a cause of neurological disease

    doi: 10.1038/nm0502-509

    Figure Lengend Snippet: Specificity of HAM/TSP IgG for CNS neurons and isolation of the neuronal antigen. a , Immunohistochemistry of human tissues. Biotinylated HAM/TSP IgG stained CNS neurons (cortex, brown reaction product) (scale bar, 100 µm). There was no staining of glia (arrowhead), dorsal root ganglion or systemic organs. A tax mAb mimicked HAM/TSP IgG staining indicated by the red stain. Neurofilament (gray) confirmed localization of tax mAb to neurons. b , Western blots of proteins derived from CNS neurons. HAM/TSP IgG showed an intense signal at 33 kD (lane 1). There was no reactivity using IgG isolated from HTLV-1 seronegative (lane 2) or HTLV-1 seropositive, neurologically asymptomatic individuals (lane 3). The tax mAb recognized the 33-kD neuronal antigen (lane 4). c , Purification and western blots of the neuronal protein following high salt extraction and centrifugation. Molecular weight markers (lane 1), precipitate (lane 2) and supernatant (lane 3). HAM/TSP IgG reacted with the supernatant (lane 3), but not the precipitate (lane 2). d , 2-D gel electrophoresis and western blot of the neuronal protein purified from the supernatant. Coomassie stain of gel (left). Western blot of the neuronal extract (right), HAM/TSP IgG reacted at 33–38 kD, pI = 9.3 (arrow).

    Article Snippet: IgG was isolated from serum or CSF by adsorption to protein A-Sepharose (Sigma) and biotinylated (biotin-N-hydroxysuccinimide, Vector, Burlingame, California).

    Techniques: Isolation, Immunohistochemistry, Staining, Western Blot, Derivative Assay, Purification, Centrifugation, Molecular Weight, Nucleic Acid Electrophoresis

    N and O-glycosylation of proteins are common post-translational modifications in Toxoplasma tachyzoites. Lysates of purified tachyzoites were incubated in the absence (Control) or presence (PNGase F) of PNGase F for 16 hours at 37°C, fractionated by SDS-PAGE on 12% (A) or 7.5% (B) gels, and transferred to nitrocellulose. Blots were probed with biotinylated lectins concanavalin-A (con-A) and Dolichos biflorus agglutinin (DBA), and with monospecific antisera to the Toxoplasma glycoprotein GAP50 and the cytoplasmic proteins MLC1 and IMC1. Arrowheads indicate the con-A-reactive parasite proteins that are PNGase F sensitive. GAP50+OS and GAP50−OS refer the fully N-glycosylated and enzymatically deglycosylated GAP50, respectively.

    Journal: Molecular and biochemical parasitology

    Article Title: N-linked glycosylation of proteins in the protozoan parasite Toxoplasma gondii

    doi: 10.1016/j.molbiopara.2007.10.012

    Figure Lengend Snippet: N and O-glycosylation of proteins are common post-translational modifications in Toxoplasma tachyzoites. Lysates of purified tachyzoites were incubated in the absence (Control) or presence (PNGase F) of PNGase F for 16 hours at 37°C, fractionated by SDS-PAGE on 12% (A) or 7.5% (B) gels, and transferred to nitrocellulose. Blots were probed with biotinylated lectins concanavalin-A (con-A) and Dolichos biflorus agglutinin (DBA), and with monospecific antisera to the Toxoplasma glycoprotein GAP50 and the cytoplasmic proteins MLC1 and IMC1. Arrowheads indicate the con-A-reactive parasite proteins that are PNGase F sensitive. GAP50+OS and GAP50−OS refer the fully N-glycosylated and enzymatically deglycosylated GAP50, respectively.

    Article Snippet: This was blocked in 3% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) and incubated for one hour with biotinylated concanavalin-A or Dolichos biflorus agglutinin (Vector, Burlingame, CA) diluted 1:2000 and 1:1000 in TBST, respectively.

    Techniques: Purification, Incubation, SDS Page

    Tranilast-induced stress alleviation decompresses tumor blood vessel improving perfusion and doxorubicin delivery in tumors. Representative images from histological analysis ( A ). Endothelial cell-specific marker CD31 (red), biotinylated tomato lectin (green) and co-expression (yellow) indicating the presence of perfused vessels, and ( B ). Expression of CD31 (red) indicating blood vessels in 4T1 and MCF10CA1a tumors. Quantification of blood vessel diameter ( C ), fraction of perfused vessels ( D ), and CD31 ( E ) area fractions in control and tranilast-treated tumors. Vessel diameter was significantly increased in both tumor models ( p = 0.038, 4T1 and p = 0.04, MCF10CA1a, n = 6–8), resulting in a significant increase in the perfused vessel fraction ( p = 0.027, 4T1 and p

    Journal: Scientific Reports

    Article Title: Tranilast-induced stress alleviation in solid tumors improves the efficacy of chemo- and nanotherapeutics in a size-independent manner

    doi: 10.1038/srep46140

    Figure Lengend Snippet: Tranilast-induced stress alleviation decompresses tumor blood vessel improving perfusion and doxorubicin delivery in tumors. Representative images from histological analysis ( A ). Endothelial cell-specific marker CD31 (red), biotinylated tomato lectin (green) and co-expression (yellow) indicating the presence of perfused vessels, and ( B ). Expression of CD31 (red) indicating blood vessels in 4T1 and MCF10CA1a tumors. Quantification of blood vessel diameter ( C ), fraction of perfused vessels ( D ), and CD31 ( E ) area fractions in control and tranilast-treated tumors. Vessel diameter was significantly increased in both tumor models ( p = 0.038, 4T1 and p = 0.04, MCF10CA1a, n = 6–8), resulting in a significant increase in the perfused vessel fraction ( p = 0.027, 4T1 and p

    Article Snippet: Next, mice were injected intracardially with 100 μl biotinylated lectin (1 mg/ml, Vector Labs) which was allowed to distribute throughout the body for 7 minutes .

    Techniques: Marker, Expressing

    Down-modulation of B7-molecules and suppression of T-cell proliferation is CTLA-4-dependent. (a) BM-derived DCs (0·5 × 10 6 /ml) were cultured either in medium alone, or with wild type CD4 +  CD25 +  T cells (2 × 10 6 /ml) (CD25 + ), CD4 +  CD25 −  T cells (CD25 − ), or a 1 : 1 mixture of T cells (CD25 +/− ) (2 × 10 6 /ml) and Con A (2·5 µg/ml). Where indicated, anti-CTLA-4 (100 µg/ml) together with Fc-receptor blocking antibody 2.4G2 (2·5 µg/ml) were included in the cultures. The cultures were harvested and the DCs analysed for cell surface expression of CD80 and CD86 after 48 hr of culture. The results are presented as percentage of the GeoMFI (CD80 = 534 and CD86 = 581) obtained from analysis of DCs grown in the absence of T cells. (b) Parallel cultures to those in (a) were assayed for proliferation after 48 hr of culture.  Ctla-4 -deficient CD4 +  CD25 +  T cells are anergic but do not down-modulate B7-molecules on DCs. (c) CD4 +  CD25 +  T cells (2·5 × 10 4 /well) from  Ctla-4 -deficient and normal (wild type) mice were cultured with DCs (1 × 10 4 /well) and anti-CD3 (1 µg/ml) or anti-CD3 + anti-CD28 (5 µg/ml) and proliferation assayed after 72 hr (d) DCs (0·5 × 10 6 /ml) were cultured for 48 hr either in medium alone or with anti-CD3 stimulated (1 µg/ml) CD4 +  CD25 +  T cells (2 × 10 6 /ml) either from wild type,  Cd28 -deficient or  Ctla-4 -deficient mice. Cells were harvested, stained and CD11c +  cells were analysed for CD80 and CD86 cell surface expression by flow cytometry. The results are presented as percentage of the GeoMFI (CD80 = 402 and CD86 = 2184) obtained from analysis of DCs grown in the absence of T cells. In (d) only age-matched wild type controls for the  Ctla-4 -deficient mice are shown, because the adult controls for the  Cd28 -deficient mice gave similar results. The data shown are representative of two to four independent experiments.

    Journal: Immunology

    Article Title: Cytotoxic T lymphocyte antigen-4-dependent down-modulation of costimulatory molecules on dendritic cells in CD4+ CD25+ regulatory T-cell-mediated suppression

    doi: 10.1111/j.1365-2567.2006.02362.x

    Figure Lengend Snippet: Down-modulation of B7-molecules and suppression of T-cell proliferation is CTLA-4-dependent. (a) BM-derived DCs (0·5 × 10 6 /ml) were cultured either in medium alone, or with wild type CD4 + CD25 + T cells (2 × 10 6 /ml) (CD25 + ), CD4 + CD25 − T cells (CD25 − ), or a 1 : 1 mixture of T cells (CD25 +/− ) (2 × 10 6 /ml) and Con A (2·5 µg/ml). Where indicated, anti-CTLA-4 (100 µg/ml) together with Fc-receptor blocking antibody 2.4G2 (2·5 µg/ml) were included in the cultures. The cultures were harvested and the DCs analysed for cell surface expression of CD80 and CD86 after 48 hr of culture. The results are presented as percentage of the GeoMFI (CD80 = 534 and CD86 = 581) obtained from analysis of DCs grown in the absence of T cells. (b) Parallel cultures to those in (a) were assayed for proliferation after 48 hr of culture. Ctla-4 -deficient CD4 + CD25 + T cells are anergic but do not down-modulate B7-molecules on DCs. (c) CD4 + CD25 + T cells (2·5 × 10 4 /well) from Ctla-4 -deficient and normal (wild type) mice were cultured with DCs (1 × 10 4 /well) and anti-CD3 (1 µg/ml) or anti-CD3 + anti-CD28 (5 µg/ml) and proliferation assayed after 72 hr (d) DCs (0·5 × 10 6 /ml) were cultured for 48 hr either in medium alone or with anti-CD3 stimulated (1 µg/ml) CD4 + CD25 + T cells (2 × 10 6 /ml) either from wild type, Cd28 -deficient or Ctla-4 -deficient mice. Cells were harvested, stained and CD11c + cells were analysed for CD80 and CD86 cell surface expression by flow cytometry. The results are presented as percentage of the GeoMFI (CD80 = 402 and CD86 = 2184) obtained from analysis of DCs grown in the absence of T cells. In (d) only age-matched wild type controls for the Ctla-4 -deficient mice are shown, because the adult controls for the Cd28 -deficient mice gave similar results. The data shown are representative of two to four independent experiments.

    Article Snippet: T cells were polyclonally stimulated by the addition of 1 µg/ml of anti-CD3 antibodies (145·2C11) or 2·5 µg/ml concanavalin A (Con A; Amersham Pharmacia, Uppsala, Sweden) to the cultures.

    Techniques: Derivative Assay, Cell Culture, Blocking Assay, Expressing, Mouse Assay, Staining, Flow Cytometry, Cytometry

    SPR sensorgrams (without background subtraction) of non-specific BSA (a) and specific Con A (b) binding to mannose functionalized chips with different underlying chemistries. C 11  substrate: 11-mercaptoundecanol, (OEG) 3 -C 11  substrate: (11-mercaptoundecyl)

    Journal: Bioconjugate chemistry

    Article Title: A Versatile Method for Functionalizing Surfaces with Bioactive Glycans

    doi: 10.1021/bc1003372

    Figure Lengend Snippet: SPR sensorgrams (without background subtraction) of non-specific BSA (a) and specific Con A (b) binding to mannose functionalized chips with different underlying chemistries. C 11 substrate: 11-mercaptoundecanol, (OEG) 3 -C 11 substrate: (11-mercaptoundecyl)

    Article Snippet: Concanavalin A (Con A) was purchased from MP Biomedicals (Solon, OH).

    Techniques: SPR Assay, Binding Assay

    SPRi shows that the response of Con A varies as a function of the nucleophile used for conjugation of mannose to the surface.

    Journal: Bioconjugate chemistry

    Article Title: A Versatile Method for Functionalizing Surfaces with Bioactive Glycans

    doi: 10.1021/bc1003372

    Figure Lengend Snippet: SPRi shows that the response of Con A varies as a function of the nucleophile used for conjugation of mannose to the surface.

    Article Snippet: Concanavalin A (Con A) was purchased from MP Biomedicals (Solon, OH).

    Techniques: Conjugation Assay

    Characterization of C. albicans and S. gordonii single- and dual-species biofilms formed on titanium using CSLM and 3D reconstruction software. Biofilms were grown for 24 h in BMM synthetic saliva in a 48-well plate containing titanium discs, and stained with Concanavalin A-Alexa Fluor ® 488 conjugate, 1 x FilmTracer™ SYPRO ® Ruby Biofilm Matrix Stain, and DAPI. Scale bar corresponds to 50 µm. Included in the figure are the xy , xz , and yz views of the resulting biofilm.

    Journal: Journal of Fungi

    Article Title: An In Vitro Model for Candida albicans–Streptococcus gordonii Biofilms on Titanium Surfaces

    doi: 10.3390/jof4020066

    Figure Lengend Snippet: Characterization of C. albicans and S. gordonii single- and dual-species biofilms formed on titanium using CSLM and 3D reconstruction software. Biofilms were grown for 24 h in BMM synthetic saliva in a 48-well plate containing titanium discs, and stained with Concanavalin A-Alexa Fluor ® 488 conjugate, 1 x FilmTracer™ SYPRO ® Ruby Biofilm Matrix Stain, and DAPI. Scale bar corresponds to 50 µm. Included in the figure are the xy , xz , and yz views of the resulting biofilm.

    Article Snippet: Biofilms on titanium were stained in a sequential order starting with 25 μg/mL Concanavalin A–Alexa Fluor® 488 conjugate (excitation/emission; 495/519) (Molecular Probes, Eugene, OR, USA) for 30 min at 37 °C, followed by 1 x FilmTracer™ SYPRO® Ruby Biofilm Matrix Stain (Molecular Probes) for 30 min at room temperature, and finally with 300 nM 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (excitation/emission; 358/461) (Molecular Probes, Eugene, OR, USA) for 10 min at 37 °C.

    Techniques: Software, Staining

    Characterization of C. albicans and S. gordonii single- and dual-species biofilms formed on titanium using confocal laser scanning microscopy (CLSM) and 3D reconstruction software. Biofilms were grown for 24 h in 1:1 v / v RPMI/THB + YE in a 48-well plate containing titanium discs, and stained with Concanavalin A-Alexa Fluor ® 488 conjugate, 1 x FilmTracer™ SYPRO ® Ruby Biofilm Matrix Stain, and DAPI. Scale bar corresponds to 50 µm. Included in the figure are the xy , xz , and yz views of the resulting biofilm.

    Journal: Journal of Fungi

    Article Title: An In Vitro Model for Candida albicans–Streptococcus gordonii Biofilms on Titanium Surfaces

    doi: 10.3390/jof4020066

    Figure Lengend Snippet: Characterization of C. albicans and S. gordonii single- and dual-species biofilms formed on titanium using confocal laser scanning microscopy (CLSM) and 3D reconstruction software. Biofilms were grown for 24 h in 1:1 v / v RPMI/THB + YE in a 48-well plate containing titanium discs, and stained with Concanavalin A-Alexa Fluor ® 488 conjugate, 1 x FilmTracer™ SYPRO ® Ruby Biofilm Matrix Stain, and DAPI. Scale bar corresponds to 50 µm. Included in the figure are the xy , xz , and yz views of the resulting biofilm.

    Article Snippet: Biofilms on titanium were stained in a sequential order starting with 25 μg/mL Concanavalin A–Alexa Fluor® 488 conjugate (excitation/emission; 495/519) (Molecular Probes, Eugene, OR, USA) for 30 min at 37 °C, followed by 1 x FilmTracer™ SYPRO® Ruby Biofilm Matrix Stain (Molecular Probes) for 30 min at room temperature, and finally with 300 nM 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (excitation/emission; 358/461) (Molecular Probes, Eugene, OR, USA) for 10 min at 37 °C.

    Techniques: Confocal Laser Scanning Microscopy, Software, Staining

    Sec13 H/− Mice Present Specific Immunological Alterations. ( a ) Analysis of immunological cell populations in Sec13 +/+ and Sec13 H/− mice. Cell subsets isolated from spleen and mesenteric lymph nodes (MLN) were analyzed by flow cytometry using CD4+ and CD8+ as phenotypic markers for T lymphocytes, CD19+ for B lymphocytes, and CD11b+CD11c- for macrophages. Results are the mean ± SD (n = 8). Intracellular cytokine production (IL-6, IL-10, and IL-12) was measured by flow cytometry in cultured cells in the presence of ConA and GolgiStop®. ( b – e ) Splenic cells from Sec13 +/+ and Sec13 H/− mice were subjected to flow cytometry to analyze frequencies of specific populations and expression of molecules. Cells were harvested and cultured for 48h in the presence of Con-A. Intracellular cytokine production was measured by flow cytometry in cultured cells in the presence of ConA and GolgiStop®. Cells were also stained with FITC-labeled anti-CD8, PercP-labeled anti-CD3 and PE-labeled anti-IFN-γ. Bar graphs are shown as mean ± SEM. ( b , c ) Cytokine production by splenic cells was measured in the supernatant by sandwich ELISA. Bar graphs are shown as mean ± SEM. ( d ) MHC I and MHC II expression was measured in CD11b+ CD11- cells isolated from mesenteric lymph nodes, spleen, and peritoneum. Graphs are representative of 6 mice/group. ( e ) Cells were stained with FITC-labeled anti-CD4, PercP-labeled anti-CD25, and PE-labeled anti-LAP. CD4+ cells and CD25+ were gated. LAP+ cells were gated within CD4+ CD25+ cells. Plots are representative of the mean of 3 mice/group. All data are representative of three independent experiments. Bar graphs are shown as mean ± SEM. Student T test was applied. *p

    Journal: Scientific Reports

    Article Title: Sec13 Regulates Expression of Specific Immune Factors Involved in Inflammation In Vivo

    doi: 10.1038/srep17655

    Figure Lengend Snippet: Sec13 H/− Mice Present Specific Immunological Alterations. ( a ) Analysis of immunological cell populations in Sec13 +/+ and Sec13 H/− mice. Cell subsets isolated from spleen and mesenteric lymph nodes (MLN) were analyzed by flow cytometry using CD4+ and CD8+ as phenotypic markers for T lymphocytes, CD19+ for B lymphocytes, and CD11b+CD11c- for macrophages. Results are the mean ± SD (n = 8). Intracellular cytokine production (IL-6, IL-10, and IL-12) was measured by flow cytometry in cultured cells in the presence of ConA and GolgiStop®. ( b – e ) Splenic cells from Sec13 +/+ and Sec13 H/− mice were subjected to flow cytometry to analyze frequencies of specific populations and expression of molecules. Cells were harvested and cultured for 48h in the presence of Con-A. Intracellular cytokine production was measured by flow cytometry in cultured cells in the presence of ConA and GolgiStop®. Cells were also stained with FITC-labeled anti-CD8, PercP-labeled anti-CD3 and PE-labeled anti-IFN-γ. Bar graphs are shown as mean ± SEM. ( b , c ) Cytokine production by splenic cells was measured in the supernatant by sandwich ELISA. Bar graphs are shown as mean ± SEM. ( d ) MHC I and MHC II expression was measured in CD11b+ CD11- cells isolated from mesenteric lymph nodes, spleen, and peritoneum. Graphs are representative of 6 mice/group. ( e ) Cells were stained with FITC-labeled anti-CD4, PercP-labeled anti-CD25, and PE-labeled anti-LAP. CD4+ cells and CD25+ were gated. LAP+ cells were gated within CD4+ CD25+ cells. Plots are representative of the mean of 3 mice/group. All data are representative of three independent experiments. Bar graphs are shown as mean ± SEM. Student T test was applied. *p

    Article Snippet: For TGF-β measurements, specific antibodies were purchased from R & D. For intracellular staining, primary cultures were stimulated with Concanavalin A (ConA) for 12 h in the presence of 10 μg/ml brefeldin A from BD Bioscience.

    Techniques: Mouse Assay, Isolation, Flow Cytometry, Cytometry, Cell Culture, Expressing, Staining, Labeling, Sandwich ELISA

    Cellular localization of FITC-labeled siRNA (green) following magnetofection (MF; A, B) and lipofection (L2000; C, D) procedures in primary fibroblasts imaged by confocal laser scanning microscopy. Both a siRNA-only control in the absence of transfection reagent (A, 2 μg siRNA; C, 1 μg siRNA) and siRNA/transfection reagent complexes (B, D) were applied. Nuclear DNA was counterstained with DAPI (blue). The cell surface marker, ConA Alexa Fluor 594 (orange-red), was used to stain the cell membrane. The last column (“merge”) shows images from the first three columns superimposed, illustrating intracellular uptake of siRNA/transfection reagent complexes (B, D) and absence of cellular uptake of FITC-siRNA alone (A, C). Co-localization of FITC-siRNA-only or siRNA/transfection reagent complexes with the cell surface did not occur.

    Journal: Biotechnic & histochemistry : official publication of the Biological Stain Commission

    Article Title: Efficient and gentle siRNA delivery by magnetofection

    doi: 10.3109/10520291003675485

    Figure Lengend Snippet: Cellular localization of FITC-labeled siRNA (green) following magnetofection (MF; A, B) and lipofection (L2000; C, D) procedures in primary fibroblasts imaged by confocal laser scanning microscopy. Both a siRNA-only control in the absence of transfection reagent (A, 2 μg siRNA; C, 1 μg siRNA) and siRNA/transfection reagent complexes (B, D) were applied. Nuclear DNA was counterstained with DAPI (blue). The cell surface marker, ConA Alexa Fluor 594 (orange-red), was used to stain the cell membrane. The last column (“merge”) shows images from the first three columns superimposed, illustrating intracellular uptake of siRNA/transfection reagent complexes (B, D) and absence of cellular uptake of FITC-siRNA alone (A, C). Co-localization of FITC-siRNA-only or siRNA/transfection reagent complexes with the cell surface did not occur.

    Article Snippet: This was followed by a direct immunostaining protocol using concanavalin A Alexa Fluor 594 conjugate for cell surface labeling (Invitrogen).

    Techniques: Labeling, Magnetofection, Confocal Laser Scanning Microscopy, Transfection, Marker, Staining

    Effect of oral administration of the  L. pentosus  strain S-PT84 on stress-induced immune compromisation of splenocytes. Splenocytes were collected from mice that were fed an AIN-93 M diet with or without S-PT84 for 1 week and then restrained. The number of splenocytes (A), splenic NK activity against target YAC-1 cells (effector:target ratio = 80:1, 40:1, and 20:1) (B), and IFN-γ (C) and IL-4 (D) production levels in splenocytes cultured with Con A (2.5 µg/ml) for 24 hr were measured as described in MATERIALS AND METHODS. Data are presented as the mean ± SE of five mice. Different letters ( a ,  b  and  c ) indicate significant differences between groups (p

    Journal: Bioscience of Microbiota, Food and Health

    Article Title: Anti-stress effect of the Lactobacillus pentosus strain S-PT84 in mice

    doi: 10.12938/bmfh.17-003

    Figure Lengend Snippet: Effect of oral administration of the L. pentosus strain S-PT84 on stress-induced immune compromisation of splenocytes. Splenocytes were collected from mice that were fed an AIN-93 M diet with or without S-PT84 for 1 week and then restrained. The number of splenocytes (A), splenic NK activity against target YAC-1 cells (effector:target ratio = 80:1, 40:1, and 20:1) (B), and IFN-γ (C) and IL-4 (D) production levels in splenocytes cultured with Con A (2.5 µg/ml) for 24 hr were measured as described in MATERIALS AND METHODS. Data are presented as the mean ± SE of five mice. Different letters ( a , b and c ) indicate significant differences between groups (p

    Article Snippet: Macrophages were cultured with lipopolysaccharide (LPS, from Escherichia coli 055; 100 ng/ml; Wako Pure Chemical industries, Osaka, Japan) for 48 hr, and splenocytes were cultured with Concanavalin A (Con A; 2.5 µg/ml, Wako) for 24 hr at 37°C.

    Techniques: Mouse Assay, Activity Assay, Cell Culture

    An automated platform for high-throughput  in situ  profiling of chromatin proteins. ( a ) AutoCUT  RUN workflow. (1) Cells or tissue are bound to Concanavalin A-coated beads, permeabilized with digitonin, and incubated with an antibody targeting a chromatin protein. (2) Samples are arrayed in a 96-well plate and (3) processed on a Biomek robot fitted with a 96-well magnetic plate for magnetic separation during washes (α), and an aluminum chiller block (β) routed to a circulating water bath (γ) for temperature control. (4) AutoCUT  RUN produces in 2 days up to 96 libraries that are ready to be pooled and sequenced. ( b ) Hierarchically clustered correlation matrix of AutoCUT  RUN profiles of histone-H3 modifications that mark active (pink) and repressed (blue) chromatin in H1 (orange) and K562 (purple) cells. Pearson correlations were calculated using the log 2  transformed values of read counts split into 500 bp bins across the genome.

    Journal: bioRxiv

    Article Title: Automated in situ profiling of chromatin modifications resolves cell types and gene regulatory programs

    doi: 10.1101/418681

    Figure Lengend Snippet: An automated platform for high-throughput in situ profiling of chromatin proteins. ( a ) AutoCUT RUN workflow. (1) Cells or tissue are bound to Concanavalin A-coated beads, permeabilized with digitonin, and incubated with an antibody targeting a chromatin protein. (2) Samples are arrayed in a 96-well plate and (3) processed on a Biomek robot fitted with a 96-well magnetic plate for magnetic separation during washes (α), and an aluminum chiller block (β) routed to a circulating water bath (γ) for temperature control. (4) AutoCUT RUN produces in 2 days up to 96 libraries that are ready to be pooled and sequenced. ( b ) Hierarchically clustered correlation matrix of AutoCUT RUN profiles of histone-H3 modifications that mark active (pink) and repressed (blue) chromatin in H1 (orange) and K562 (purple) cells. Pearson correlations were calculated using the log 2 transformed values of read counts split into 500 bp bins across the genome.

    Article Snippet: Concanavalin A-coated magnetic beads (Bangs Laboratories, ca. no. BP531).

    Techniques: High Throughput Screening Assay, In Situ, Incubation, Blocking Assay, Transformation Assay

    Analysis of UT-A1 glycan sialylation in PKC- α  knockout mouse. Kidney IM tissues were dissected from C57B6 wild-type (WT) or PKC- α  knockout mice and processed for lipid raft isolation by 5%–40% sucrose gradient ultracentrifugation. Lipid raft fractions (fractions 2–5) were collected and used for a lectin pull-down assay. UT-A1 proteins from (A) cell membrane lipid rafts and (B) lectin-precipitated samples were examined by Western blot with UT-A1 antibody. Con A, concanavalin A; IB, immunoblot; MAA, maackia amurensis agglutinin; Tomato, lycopersicum esculentum lectin; WGA, wheat germ agglutinin.

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Activation of Protein Kinase C-α and Src Kinase Increases Urea Transporter A1 α-2, 6 Sialylation

    doi: 10.1681/ASN.2014010026

    Figure Lengend Snippet: Analysis of UT-A1 glycan sialylation in PKC- α knockout mouse. Kidney IM tissues were dissected from C57B6 wild-type (WT) or PKC- α knockout mice and processed for lipid raft isolation by 5%–40% sucrose gradient ultracentrifugation. Lipid raft fractions (fractions 2–5) were collected and used for a lectin pull-down assay. UT-A1 proteins from (A) cell membrane lipid rafts and (B) lectin-precipitated samples were examined by Western blot with UT-A1 antibody. Con A, concanavalin A; IB, immunoblot; MAA, maackia amurensis agglutinin; Tomato, lycopersicum esculentum lectin; WGA, wheat germ agglutinin.

    Article Snippet: Agarose-bound concanavalin A, wheat germ agglutinin, SNA, GNL, datura stramonium lectin, phaseolus vulgaris leucoagglutinin, and Tomato lectin (Lycopersicum esculentum lectin) were purchased from Vector Laboratories (Burlingame, CA).

    Techniques: Knock-Out, Mouse Assay, Isolation, Pull Down Assay, Western Blot, Whole Genome Amplification

    PKC activator increases UT-A1 glycan sialylation. (A) UT-A1 MDCK cells were treated with 2  µ M PDBu for 24 hours. The cells were lysed in RIPA buffer, equal amounts of total lysates were incubated with agarose-conjugated lectins, and then, they were immunoblotted with antibody to UT-A1. (B) Kidney IMCD suspensions prepared from Sprague–Dawley rats were treated with 2  µ M PDBu for 6 hours. The cell membrane lipid rafts were prepared by a 5%–40% sucrose gradient ultracentrifugation. Fractions 2–5 were collected for lectin pull-down assay with different lectins. The lectin-precipitated samples were then analyzed for UT-A1 protein expression by Western blot. The bar graph shows band densities as fold of control (means±SDs;  n =3). Con A, concanavalin A; Ctrl, control; DSL, datura stramonium lectin; IB, immunoblot; PHA, phaseolus vulgaris leucoagglutinin; Tomato, lycopersicum esculentum lectin; WGA, wheat germ agglutinin. ** P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Activation of Protein Kinase C-α and Src Kinase Increases Urea Transporter A1 α-2, 6 Sialylation

    doi: 10.1681/ASN.2014010026

    Figure Lengend Snippet: PKC activator increases UT-A1 glycan sialylation. (A) UT-A1 MDCK cells were treated with 2 µ M PDBu for 24 hours. The cells were lysed in RIPA buffer, equal amounts of total lysates were incubated with agarose-conjugated lectins, and then, they were immunoblotted with antibody to UT-A1. (B) Kidney IMCD suspensions prepared from Sprague–Dawley rats were treated with 2 µ M PDBu for 6 hours. The cell membrane lipid rafts were prepared by a 5%–40% sucrose gradient ultracentrifugation. Fractions 2–5 were collected for lectin pull-down assay with different lectins. The lectin-precipitated samples were then analyzed for UT-A1 protein expression by Western blot. The bar graph shows band densities as fold of control (means±SDs; n =3). Con A, concanavalin A; Ctrl, control; DSL, datura stramonium lectin; IB, immunoblot; PHA, phaseolus vulgaris leucoagglutinin; Tomato, lycopersicum esculentum lectin; WGA, wheat germ agglutinin. ** P

    Article Snippet: Agarose-bound concanavalin A, wheat germ agglutinin, SNA, GNL, datura stramonium lectin, phaseolus vulgaris leucoagglutinin, and Tomato lectin (Lycopersicum esculentum lectin) were purchased from Vector Laboratories (Burlingame, CA).

    Techniques: Incubation, Pull Down Assay, Expressing, Western Blot, Whole Genome Amplification

    The effect of R164Q mutation on the signal transduction and cell surface expression of PKR2. A , R164Q mutation disrupted the signal transduction of PKR2 as assayed with an aequorin-based assay. B , both WT and R164Q-PKR2 were presented on the cell surface. The Alexa Fluor 594-conjugated concanavalin A was used to label the membrane. The fluorescence was monitored with a confocal microscope. Scale bar: 10 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: Disease-causing Mutation in PKR2 Receptor Reveals a Critical Role of Positive Charges in the Second Intracellular Loop for G-protein Coupling and Receptor Trafficking

    doi: 10.1074/jbc.M111.223784

    Figure Lengend Snippet: The effect of R164Q mutation on the signal transduction and cell surface expression of PKR2. A , R164Q mutation disrupted the signal transduction of PKR2 as assayed with an aequorin-based assay. B , both WT and R164Q-PKR2 were presented on the cell surface. The Alexa Fluor 594-conjugated concanavalin A was used to label the membrane. The fluorescence was monitored with a confocal microscope. Scale bar: 10 μm.

    Article Snippet: Alexa Fluor 594-conjugated concanavalin A (Invitrogen) was applied to label the membrane.

    Techniques: Mutagenesis, Transduction, Expressing, Fluorescence, Microscopy

    Principle and proof of inducible speckle imaging. (a) A single transverse mode laser beam is scattered by a diffuser before the microscope objective, creating a pattern composed of speckles with a characteristic size (correlation length) at the diffraction limit. This high-contrast pattern is generated at all planes: at, before, and after the objective’s focal plane. The beam induces a heterogeneous photoswitch pattern across the sample. (b) A PSF volume comprises a large number of potentially tagged slots. In FSM, slots are randomly occupied, leading to faint density fluctuations at the PSF scale. Increased fluctuations can be induced by narrowing the PSF or, conversely, by clustering fluorescent molecules, as does ISI, to emulate PSF-sized slots. (c) Comparison between simulated and observed fluorescence pattern structure after an ISI pulse on a uniform fluorescent layer (concanavalin A–conjugated Alexa Fluor 488) at different bleaching strengths (γ). Bars, 2 µm. (d) ISI on GFP–α-tubulin in a fixed Drosophila S2 mitotic cell. To produce a roughly uniform fluorophore pool, microtubules were depolymerized using colchicine. Bars: 5 µm; (inset) 1 µm.

    Journal: The Journal of Cell Biology

    Article Title: Inducible fluorescent speckle microscopy

    doi: 10.1083/jcb.201506128

    Figure Lengend Snippet: Principle and proof of inducible speckle imaging. (a) A single transverse mode laser beam is scattered by a diffuser before the microscope objective, creating a pattern composed of speckles with a characteristic size (correlation length) at the diffraction limit. This high-contrast pattern is generated at all planes: at, before, and after the objective’s focal plane. The beam induces a heterogeneous photoswitch pattern across the sample. (b) A PSF volume comprises a large number of potentially tagged slots. In FSM, slots are randomly occupied, leading to faint density fluctuations at the PSF scale. Increased fluctuations can be induced by narrowing the PSF or, conversely, by clustering fluorescent molecules, as does ISI, to emulate PSF-sized slots. (c) Comparison between simulated and observed fluorescence pattern structure after an ISI pulse on a uniform fluorescent layer (concanavalin A–conjugated Alexa Fluor 488) at different bleaching strengths (γ). Bars, 2 µm. (d) ISI on GFP–α-tubulin in a fixed Drosophila S2 mitotic cell. To produce a roughly uniform fluorophore pool, microtubules were depolymerized using colchicine. Bars: 5 µm; (inset) 1 µm.

    Article Snippet: Thin fluorescent layers (experiments in and ) were prepared by coating coverslips with Alexa Fluor 488–conjugated concanavalin A (Molecular Probes).

    Techniques: Imaging, Microscopy, Generated, Fluorescence

    Confocal laser microscopy images of MDA-MB-231 breast cancer cells incubated for 8 hours with PaxOG-eNPs. ( top left ) Intracellular Oregon Green® 488 Taxol signal detected using 488 nm laser. ( top right ) Concanavalin A-Alexa Fluor 647 signal detected using 633 nm laser and marking the plasma membrane in red. ( bottom left ) Differential interference contrast image. ( bottom right ) Composite image showing intracellular delivery of the Oregon Green® 488 Taxol to the cell cytoplasm. Scale bar = 10 μm.

    Journal: Biomacromolecules

    Article Title: In Vitro Activity of Paclitaxel-Loaded Polymeric Expansile Nanoparticles in Breast Cancer Cells

    doi: 10.1021/bm400434h

    Figure Lengend Snippet: Confocal laser microscopy images of MDA-MB-231 breast cancer cells incubated for 8 hours with PaxOG-eNPs. ( top left ) Intracellular Oregon Green® 488 Taxol signal detected using 488 nm laser. ( top right ) Concanavalin A-Alexa Fluor 647 signal detected using 633 nm laser and marking the plasma membrane in red. ( bottom left ) Differential interference contrast image. ( bottom right ) Composite image showing intracellular delivery of the Oregon Green® 488 Taxol to the cell cytoplasm. Scale bar = 10 μm.

    Article Snippet: The coverslips were washed with PBS (2 mL × 1) and PBS containing calcium and magnesium (complete PBS; 2 mL × 1) and then incubated with 50 μg/mL concanavalin A-Alexa Fluor 647 conjugate (Invitrogen) in PBS (1 mL) for 5 min in order to identify the cell membrane.

    Techniques: Microscopy, Multiple Displacement Amplification, Incubation

    Comparison of N-glycan profiles in HepG2 cells treated with and without 2FF. ( A ) MS spectrum of the permethylated glycans from the cells treated with DMSO only (upper) and DMSO containing 2FF (lower). ( B ) Percentages of fucosylated complex glycans in total complex glycans from HepG2 cells. The values were calculated from peak areas in the range of m/z 1,500-3,500 of the MS spectra. ( C ) HepG2 cells were cultured with 2FF for 3 days at different concentrations as indicated, and then equal amounts of cell lysates were probed with Con A, which specifically recognizes alpha-linked mannose and glucose; WGA, which selectively binds to N-Acetyl glucosamine (GlcNAc) and SNA, which specifically recognizes α 2, 6 sialylation. GAPDH was used as a loading control.

    Journal: Scientific Reports

    Article Title: Inhibition of fucosylation by 2-fluorofucose suppresses human liver cancer HepG2 cell proliferation and migration as well as tumor formation

    doi: 10.1038/s41598-017-11911-9

    Figure Lengend Snippet: Comparison of N-glycan profiles in HepG2 cells treated with and without 2FF. ( A ) MS spectrum of the permethylated glycans from the cells treated with DMSO only (upper) and DMSO containing 2FF (lower). ( B ) Percentages of fucosylated complex glycans in total complex glycans from HepG2 cells. The values were calculated from peak areas in the range of m/z 1,500-3,500 of the MS spectra. ( C ) HepG2 cells were cultured with 2FF for 3 days at different concentrations as indicated, and then equal amounts of cell lysates were probed with Con A, which specifically recognizes alpha-linked mannose and glucose; WGA, which selectively binds to N-Acetyl glucosamine (GlcNAc) and SNA, which specifically recognizes α 2, 6 sialylation. GAPDH was used as a loading control.

    Article Snippet: Biotinylated aleuria aurantia lectin (AAL), concanavalin A (ConA) and wheat germ agglutinin (WGA) were purchased from Seikagaku Crop (Tokyo, Japan).

    Techniques: Mass Spectrometry, Cell Culture, Whole Genome Amplification