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  • 95
    Vector Laboratories biotinylated concanavalin a
    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 <t>biotinylated</t> lectins <t>concanavalin-A</t> (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.
    Biotinylated Concanavalin A, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 95/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore concanavalin a con a
    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 <t>biotinylated</t> lectins <t>concanavalin-A</t> (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.
    Concanavalin A Con A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bangalore Genei concanavalin a con a
    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 <t>biotinylated</t> lectins <t>concanavalin-A</t> (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.
    Concanavalin A Con A, supplied by Bangalore Genei, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    HiMedia Laboratories concanavalin a con a
    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 <t>biotinylated</t> lectins <t>concanavalin-A</t> (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.
    Concanavalin A Con A, supplied by HiMedia Laboratories, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    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: 95/100, based on 412 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore concanavalin a
    N -Glycosylated proteins with high-mannose oligosaccharide chains are much less abundant in the tonoplast than in the total membrane fraction. Microsomal fractions (M) from Arabidopsis protoplasts or purified vacuoles were analyzed by SDS–PAGE and protein blot followed by incubation with <t>concanavalin</t> A conjugated to peroxidase. Numbers on the left indicate the positions of molecular mass markers, in kDa.
    Concanavalin A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 14549 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Vector Laboratories concanavalin a con a agarose
    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.
    Concanavalin A Con A Agarose, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 94/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    InvivoGen con a injection
    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.
    Con A Injection, supplied by InvivoGen, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    GE Healthcare concanavalin a con a
    Induction of IFNγ-producing antigen specific CD8 +  T cells following i.d. administrations of WT1(peptide)-PSNPs candidates in HLA-A2.1/Kb mice. WT1 derived peptides (WT1A, WT1B, WT1C, WT1D, and WT1E) were covalently conjugated to PSNPs to constitute PSNPs vaccine formulations (containing 0.5 mg/ml of each peptide in each of the conjugation mix). Mice were immunized 3 times with each formulation (100 μl or 50 μg (including both conjugated and non-conjugated peptide)/injection) intradermally, 10 days apart. 11 days after the last immunization, antigen specific T cell responses were evaluated by IFN γ ELISpot assay upon stimulations with WT1 peptides (5 μg/ml) or controls (media alone or Con A). Each condition was tested in triplicate on splenocytes from individual mouse ( n  = 4). Results are expressed as stimulation index (SI) of the SFU over the background (media alone) ± SD ( n  = 4 individual mice). Two-way ANOVA analysis indicated the significance of WT1A and WT1B peptide processing in the WT1peptide-PSNPs formulations.  * p
    Concanavalin A Con A, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 88/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ICN Biomedicals concanavalin a con a
    Induction of IFNγ-producing antigen specific CD8 +  T cells following i.d. administrations of WT1(peptide)-PSNPs candidates in HLA-A2.1/Kb mice. WT1 derived peptides (WT1A, WT1B, WT1C, WT1D, and WT1E) were covalently conjugated to PSNPs to constitute PSNPs vaccine formulations (containing 0.5 mg/ml of each peptide in each of the conjugation mix). Mice were immunized 3 times with each formulation (100 μl or 50 μg (including both conjugated and non-conjugated peptide)/injection) intradermally, 10 days apart. 11 days after the last immunization, antigen specific T cell responses were evaluated by IFN γ ELISpot assay upon stimulations with WT1 peptides (5 μg/ml) or controls (media alone or Con A). Each condition was tested in triplicate on splenocytes from individual mouse ( n  = 4). Results are expressed as stimulation index (SI) of the SFU over the background (media alone) ± SD ( n  = 4 individual mice). Two-way ANOVA analysis indicated the significance of WT1A and WT1B peptide processing in the WT1peptide-PSNPs formulations.  * p
    Concanavalin A Con A, supplied by ICN Biomedicals, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Vector Laboratories fitc concanavalin a
    Imaging of purified tissue cysts A–C Assembly of the Cytospin holders to pellet the purified tissue cysts onto the slide surface. A . A glass slide and hole punched filter card are placed into the stainless steel clip holder. B . A sample funnel is placed into the holder such that the base of funnel aligns with the lower hole punch on the filter card. C . Assembled Cytospin holders. D–E . Cytospin holders are placed in the centrifuge and the sample added to the funnels using a micropipettor. F . Following centrifugation and the disassembly of the holder the slides are placed in a Coplin jar with fixative. G–I Imaging of a tissue cyst with Dolichos biflorus <t>lectin</t> (DBA-green) ( G ) and <t>Concanavalin</t> A (ConA-Red) ( H ) acquired on a Zeiss Axiophot stand using a 100× 1.4NA objective. The merged image ( I ) clearly shows the differential distribution of the glycans recognized by these lectins within the tissue cyst.
    Fitc Concanavalin A, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    Generating an infectious clone of ZIKV PRVABC59 and nonglycosylated mutants. (A) An infectious clone of ZIKV strain PRVABC59 was generated using a system that divides the viral genome into 4 fragments flanked by the indicated restriction endonuclease sites. Numbers above fragments indicate nucleotide position in the viral genome. T7 promoter and a hepatitis delta virus (HDV) ribozyme sequences flank the genome. (B) Sequence chromatograms of E protein glycosylation site of WT, N154Q, or T156I clone. (C) Vero cells were infected at an MOI of 0.01 with ZIKV PRVABC59 isolate, WT clone, N154Q mutant, or T156I mutant. Viruses in culture supernatants were titrated by focus-forming assay. Data shown are the mean values ± SEM of 9 samples from 3 independent experiments. (D and E) E proteins were immunoprecipitated with MAb 1M7 from lysates of Vero cells infected with ZIKV PRVABC59 isolate, WT clone, N154Q mutant, or T156I mutant. (D) Lysates were treated with PNGase F, separated by nonreducing SDS-PAGE, and probed with MAb 4G2. (E) Lysates were separated by nonreducing SDS-PAGE and probed with biotinylated lectin concanavalin A.

    Journal: Journal of Virology

    Article Title: Envelope Protein Glycosylation Mediates Zika Virus Pathogenesis

    doi: 10.1128/JVI.00113-19

    Figure Lengend Snippet: Generating an infectious clone of ZIKV PRVABC59 and nonglycosylated mutants. (A) An infectious clone of ZIKV strain PRVABC59 was generated using a system that divides the viral genome into 4 fragments flanked by the indicated restriction endonuclease sites. Numbers above fragments indicate nucleotide position in the viral genome. T7 promoter and a hepatitis delta virus (HDV) ribozyme sequences flank the genome. (B) Sequence chromatograms of E protein glycosylation site of WT, N154Q, or T156I clone. (C) Vero cells were infected at an MOI of 0.01 with ZIKV PRVABC59 isolate, WT clone, N154Q mutant, or T156I mutant. Viruses in culture supernatants were titrated by focus-forming assay. Data shown are the mean values ± SEM of 9 samples from 3 independent experiments. (D and E) E proteins were immunoprecipitated with MAb 1M7 from lysates of Vero cells infected with ZIKV PRVABC59 isolate, WT clone, N154Q mutant, or T156I mutant. (D) Lysates were treated with PNGase F, separated by nonreducing SDS-PAGE, and probed with MAb 4G2. (E) Lysates were separated by nonreducing SDS-PAGE and probed with biotinylated lectin concanavalin A.

    Article Snippet: For lectin blots, glycosylated E was detected with the biotinylated lectin concanavalin A (ConA; Vector Laboratories), followed by HRP-conjugated streptavidin.

    Techniques: Generated, Sequencing, Infection, Mutagenesis, Focus Forming Assay, Immunoprecipitation, SDS Page

    E glycosylation is not required for ZIKV replication. (A) ZIKV envelope protein, depicting the nucleotide and amino acid residues of the glycosylation site, and N154Q mutation. (B) Sequence chromatograms of E protein glycosylation site of wild-type (WT) and N154Q viruses. (C) Vero cells were infected at an MOI of 0.01 with ZIKV H/PF/2013 isolate, WT clone, or N154Q mutant. Viruses in culture supernatants were titrated by focus-forming assay. Data shown are the mean values ± standard errors of the means (SEM) of 9 samples from 3 independent experiments. (D and E) E proteins were immunoprecipitated with MAb 1M7 from lysates of Vero cells infected with ZIKV H/PF/2013 isolate, WT clone, N154Q mutant, or DENV. (D) Lysates were treated with PNGase F, separated by nonreducing SDS-PAGE, and probed with MAb 4G2. (E) Lysates were separated by nonreducing SDS-PAGE and probed with biotinylated concanavalin A to detect glycans. FT, flow-through; IP, immunoprecipitate.

    Journal: Journal of Virology

    Article Title: Envelope Protein Glycosylation Mediates Zika Virus Pathogenesis

    doi: 10.1128/JVI.00113-19

    Figure Lengend Snippet: E glycosylation is not required for ZIKV replication. (A) ZIKV envelope protein, depicting the nucleotide and amino acid residues of the glycosylation site, and N154Q mutation. (B) Sequence chromatograms of E protein glycosylation site of wild-type (WT) and N154Q viruses. (C) Vero cells were infected at an MOI of 0.01 with ZIKV H/PF/2013 isolate, WT clone, or N154Q mutant. Viruses in culture supernatants were titrated by focus-forming assay. Data shown are the mean values ± standard errors of the means (SEM) of 9 samples from 3 independent experiments. (D and E) E proteins were immunoprecipitated with MAb 1M7 from lysates of Vero cells infected with ZIKV H/PF/2013 isolate, WT clone, N154Q mutant, or DENV. (D) Lysates were treated with PNGase F, separated by nonreducing SDS-PAGE, and probed with MAb 4G2. (E) Lysates were separated by nonreducing SDS-PAGE and probed with biotinylated concanavalin A to detect glycans. FT, flow-through; IP, immunoprecipitate.

    Article Snippet: For lectin blots, glycosylated E was detected with the biotinylated lectin concanavalin A (ConA; Vector Laboratories), followed by HRP-conjugated streptavidin.

    Techniques: Mutagenesis, Sequencing, Infection, Focus Forming Assay, Immunoprecipitation, SDS Page, Flow Cytometry

    Results from fluorescence-radioactivity correlation studies. Data from the biotinylated ConA radioimmunoassay () were converted from units of CPM to site density using the linear regression from a standard curve generated from known amounts of [

    Journal: ACS applied materials & interfaces

    Article Title: Quantitative Photochemical Immobilization of Biomolecules on Planar and Corrugated Substrates: A Versatile Strategy for Creating Functional Biointerfaces

    doi: 10.1021/am2009597

    Figure Lengend Snippet: Results from fluorescence-radioactivity correlation studies. Data from the biotinylated ConA radioimmunoassay () were converted from units of CPM to site density using the linear regression from a standard curve generated from known amounts of [

    Article Snippet: The following biomolecules were used in photoimmobilization studies: biotinylated Concanavalin A (ConA-biotin), purchased from Vector Laboratories (Burlingame, CA); mannan isolated from Saccharomyces cerevisiae; recombinant human P-selectin (CHO cell-derived), purchased from R & D Systems (Minneapolis, MN); and fibronectin (FN), purchased from Invitrogen (Carlsbad, CA).

    Techniques: Fluorescence, Radioactivity, RIA Assay, Generated

    Protein loading is positively correlated with UV exposure time. Biotinylated ConA (5 μg/mL) was uniformly photopatterned onto BP-modified substrates for 5, 15, 30, 60, or 120 sec. The signal from subsequent (A) fluorescence analysis and (B) radioimmunoassays

    Journal: ACS applied materials & interfaces

    Article Title: Quantitative Photochemical Immobilization of Biomolecules on Planar and Corrugated Substrates: A Versatile Strategy for Creating Functional Biointerfaces

    doi: 10.1021/am2009597

    Figure Lengend Snippet: Protein loading is positively correlated with UV exposure time. Biotinylated ConA (5 μg/mL) was uniformly photopatterned onto BP-modified substrates for 5, 15, 30, 60, or 120 sec. The signal from subsequent (A) fluorescence analysis and (B) radioimmunoassays

    Article Snippet: The following biomolecules were used in photoimmobilization studies: biotinylated Concanavalin A (ConA-biotin), purchased from Vector Laboratories (Burlingame, CA); mannan isolated from Saccharomyces cerevisiae; recombinant human P-selectin (CHO cell-derived), purchased from R & D Systems (Minneapolis, MN); and fibronectin (FN), purchased from Invitrogen (Carlsbad, CA).

    Techniques: Modification, Size-exclusion Chromatography, Fluorescence

    Glass substrates were etched to generate channels 100 μm wide and 35 μm deep (A and B), followed by functionalization with BP. Biotinylated ConA was photoimmobilized using the same photomask used for etching rotated 90° and imaged

    Journal: ACS applied materials & interfaces

    Article Title: Quantitative Photochemical Immobilization of Biomolecules on Planar and Corrugated Substrates: A Versatile Strategy for Creating Functional Biointerfaces

    doi: 10.1021/am2009597

    Figure Lengend Snippet: Glass substrates were etched to generate channels 100 μm wide and 35 μm deep (A and B), followed by functionalization with BP. Biotinylated ConA was photoimmobilized using the same photomask used for etching rotated 90° and imaged

    Article Snippet: The following biomolecules were used in photoimmobilization studies: biotinylated Concanavalin A (ConA-biotin), purchased from Vector Laboratories (Burlingame, CA); mannan isolated from Saccharomyces cerevisiae; recombinant human P-selectin (CHO cell-derived), purchased from R & D Systems (Minneapolis, MN); and fibronectin (FN), purchased from Invitrogen (Carlsbad, CA).

    Techniques:

    One-component and three-component patterns of biomolecules on BP-modified substrates. (A) Photoimmobilization of biotinylated ConA in the “Illinois logo” pattern, visualized with fluorescently labeled streptavidin. (B) Three-component

    Journal: ACS applied materials & interfaces

    Article Title: Quantitative Photochemical Immobilization of Biomolecules on Planar and Corrugated Substrates: A Versatile Strategy for Creating Functional Biointerfaces

    doi: 10.1021/am2009597

    Figure Lengend Snippet: One-component and three-component patterns of biomolecules on BP-modified substrates. (A) Photoimmobilization of biotinylated ConA in the “Illinois logo” pattern, visualized with fluorescently labeled streptavidin. (B) Three-component

    Article Snippet: The following biomolecules were used in photoimmobilization studies: biotinylated Concanavalin A (ConA-biotin), purchased from Vector Laboratories (Burlingame, CA); mannan isolated from Saccharomyces cerevisiae; recombinant human P-selectin (CHO cell-derived), purchased from R & D Systems (Minneapolis, MN); and fibronectin (FN), purchased from Invitrogen (Carlsbad, CA).

    Techniques: Modification, Labeling

    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

    Optical microscope fluorescence representative images of E. hirae ATCC 10541 biofilms at 48 h on polystyrene at 20 °C ( a ) and 37 °C ( b ) on stainless steel coupon at 20 °C ( c ) and 37 °C ( d ) by Con-A staining for detecting α glucans in matrix formation. Original magnification ×1000

    Journal: Environmental Health and Preventive Medicine

    Article Title: Enterococcus hirae biofilm formation on hospital material surfaces and effect of new biocides

    doi: 10.1186/s12199-017-0670-3

    Figure Lengend Snippet: Optical microscope fluorescence representative images of E. hirae ATCC 10541 biofilms at 48 h on polystyrene at 20 °C ( a ) and 37 °C ( b ) on stainless steel coupon at 20 °C ( c ) and 37 °C ( d ) by Con-A staining for detecting α glucans in matrix formation. Original magnification ×1000

    Article Snippet: Concanavalin A assay To visualize the extracellular polymeric substance (EPS) matrix of the produced biofilms, rhodamine-labeled Concanavalin A (rhodamine-conA) (Vector Laboratories, Burlingame, CA, USA), which specifically binds to d-(+)-glucose and d-(+)-mannose groups on EPS, was used.

    Techniques: Microscopy, Fluorescence, Staining

    ( a ) GFAP is found along the GCL, and Müller cell processes are seen to project towards the plexiform layer. Representative images of GFAP expression (red) in transverse sections of eyes from 6-week-old  ob − / ob −  and  ob + / ob +  mice reveal elevated GFAP expression in  ob − / ob −  mice along the GCL (white arrowheads) and OPL (yellow arrowheads), suggesting gliosis. Scale bar, 100 μm. ( b ) In the retinal whole-mounts, there were more GFAP-labelled Müller cells (green) in the primary plexus layer in  ob − / ob −  than  ob + / ob +  mice. Gliosis by upregulated Müller cells reflects inflammation of the nervous system. Scale bar, 50 μm. ( c ) In the same layer of retinal whole-mounts, the IBA-1-labelled macrophages (green) were more abundant, amoeboid and activated in  ob − / ob −  mice. Scale bars, 150 μm. ( d ) Graph of the number of Rho-Con A-labelled leucocytes in the main retinal blood vessels in each eye, in 6- and 20-week-old  ob + / ob +  and  ob − / ob −  mice. In both age groups, there were more leucocytes in the eyes of  ob − / ob −  than  ob + / ob +  mice. This was significant at both 6 weeks ( n  = 6 eyes) and 20 weeks ( n  = 7 eyes); * p

    Journal: Diabetologia

    Article Title: BTBR ob/ob mouse model of type 2 diabetes exhibits early loss of retinal function and retinal inflammation followed by late vascular changes

    doi: 10.1007/s00125-018-4696-x

    Figure Lengend Snippet: ( a ) GFAP is found along the GCL, and Müller cell processes are seen to project towards the plexiform layer. Representative images of GFAP expression (red) in transverse sections of eyes from 6-week-old ob − / ob − and ob + / ob + mice reveal elevated GFAP expression in ob − / ob − mice along the GCL (white arrowheads) and OPL (yellow arrowheads), suggesting gliosis. Scale bar, 100 μm. ( b ) In the retinal whole-mounts, there were more GFAP-labelled Müller cells (green) in the primary plexus layer in ob − / ob − than ob + / ob + mice. Gliosis by upregulated Müller cells reflects inflammation of the nervous system. Scale bar, 50 μm. ( c ) In the same layer of retinal whole-mounts, the IBA-1-labelled macrophages (green) were more abundant, amoeboid and activated in ob − / ob − mice. Scale bars, 150 μm. ( d ) Graph of the number of Rho-Con A-labelled leucocytes in the main retinal blood vessels in each eye, in 6- and 20-week-old ob + / ob + and ob − / ob − mice. In both age groups, there were more leucocytes in the eyes of ob − / ob − than ob + / ob + mice. This was significant at both 6 weeks ( n  = 6 eyes) and 20 weeks ( n  = 7 eyes); * p

    Article Snippet: After inducing deep anaesthesia with 200 mg/kg pentobarbital sodium (Euthatal, Merial, Harlow, UK), the mice were cardiac perfused with rhodamine-conjugated concanavalin A (Rho-Con A; Vector Laboratories), followed by PBS.

    Techniques: Expressing, Mouse Assay

    Exacerbated retinal leukostasis in APN-KO mice. A, Representative photographs of retinal vasculature and adherent leukocytes (arrows) labeled with FITC-conjugated Con A lectin in WT (left) and APN-KO (right) mice at P15. B, Quantitative analysis of leukocyte

    Journal: Circulation research

    Article Title: Adiponectin Suppresses Pathological Microvessel Formation in Retina Through Modulation of Tumor Necrosis Factor-α Expression

    doi: 10.1161/CIRCRESAHA.109.194506

    Figure Lengend Snippet: Exacerbated retinal leukostasis in APN-KO mice. A, Representative photographs of retinal vasculature and adherent leukocytes (arrows) labeled with FITC-conjugated Con A lectin in WT (left) and APN-KO (right) mice at P15. B, Quantitative analysis of leukocyte

    Article Snippet: The retinal vasculature and adherent leukocytes were labeled with FITC-conjugated concanavalin A (Con A) lectin (Vector laboratory)., In brief, mice were perfused with PBS to remove the erythrocytes and nonadherent leukocytes in the retinal vasculature and then perfused with FITC–Con A lectin, followed by perfusion with PBS to remove unbound Con A lectin (n=12 to 20 for each experiment).

    Techniques: Mouse Assay, Labeling

    A significant fraction of 6AzGlc-dependent labeling is O-linked. (A) Known O -GlcNAcylated proteins are labeled by 6AzGlc. H1299 cells were treated with either Ac 4 6AzGlc (200 μ M) or DMSO for 16 h, followed by CuAAC with a cleavable alkyne-biotin tag. After enrichment on streptavidin beads, the labeled proteins were eluted and visualized by Western blotting. The nonglycosylated protein β -actin is a negative control. (B) A notable fraction of 6AzGlc-dependent signal is sensitive to β -elimination. NIH3T3 cells were treated with either Ac 4 6AzGlc (200 μ M) or DMSO vehicle for 16 h, followed by CuAAC with alkyne-biotin, separation by SDS-PAGE and transfer to a PVDF membrane. The indicated membranes were then treated for 24 h with either H 2 O or 55 mM NaOH before analysis by streptavidin or Western blotting. (C) 6AzGlc is not incorporated into N-linked glycans. NIH3T3 cells were treated with either Ac 4 6AzGlc (200 μ M) or DMSO vehicle for 16 h. The corresponding cell lysates were then incubated with either PNGase-F or H 2 O vehicle as indicated before CuAAC with alkyne TAMRA and analysis by in-gel fluorescence. A fraction of the treated lysate was separated before CuAAC and analyzed by Lectin blotting with Concanavalin A (ConA).

    Journal: Journal of the American Chemical Society

    Article Title: The Metabolic Chemical Reporter 6‑Azido-6-deoxy-glucose Further Reveals the Substrate Promiscuity of O‑GlcNAc Transferase and Catalyzes the Discovery of Intracellular Protein Modification by O‑Glucose

    doi: 10.1021/jacs.7b13488

    Figure Lengend Snippet: A significant fraction of 6AzGlc-dependent labeling is O-linked. (A) Known O -GlcNAcylated proteins are labeled by 6AzGlc. H1299 cells were treated with either Ac 4 6AzGlc (200 μ M) or DMSO for 16 h, followed by CuAAC with a cleavable alkyne-biotin tag. After enrichment on streptavidin beads, the labeled proteins were eluted and visualized by Western blotting. The nonglycosylated protein β -actin is a negative control. (B) A notable fraction of 6AzGlc-dependent signal is sensitive to β -elimination. NIH3T3 cells were treated with either Ac 4 6AzGlc (200 μ M) or DMSO vehicle for 16 h, followed by CuAAC with alkyne-biotin, separation by SDS-PAGE and transfer to a PVDF membrane. The indicated membranes were then treated for 24 h with either H 2 O or 55 mM NaOH before analysis by streptavidin or Western blotting. (C) 6AzGlc is not incorporated into N-linked glycans. NIH3T3 cells were treated with either Ac 4 6AzGlc (200 μ M) or DMSO vehicle for 16 h. The corresponding cell lysates were then incubated with either PNGase-F or H 2 O vehicle as indicated before CuAAC with alkyne TAMRA and analysis by in-gel fluorescence. A fraction of the treated lysate was separated before CuAAC and analyzed by Lectin blotting with Concanavalin A (ConA).

    Article Snippet: The blots washed three times in TBST for 5 min and incubated with biotin-conjugated Concanavalin A (Vector Lab), diluted 1:1000 in TBST, for 1 h. The blot was then washed 3× with TBST for 10 min. Then the blot was then incubated with Strep-HRP at 1:1000 in blocking buffer for 1 h. After being washed 3× with TBST for 10 min, the blot was developed using ECL reagents.

    Techniques: Labeling, Western Blot, Negative Control, SDS Page, Incubation, Fluorescence

    Work flow illustrating the steps involved in the proteomic analysis of OA synovial fluid. OA synovial fluid samples were pooled and subjected to depletion of abundant proteins by MARS-6 LC column and lectin affinity chromatography using three different lectins (Concanavalin A, wheat germ agglutinin and jacalin). The depleted fraction was then subjected to SDS-PAGE, SCX and OFFGEL fractionation. The lectin enriched fraction was subjected to SDS-PAGE analysis and SCX fractionation. All fractions were analyzed on LTQ-Orbitrap Velos mass spectrometer. Sequest and Mascot algorithms were used to perform database searches. Subsequently, gene ontology-based functional characterization of the identified synovial fluid proteins was carried out. Further, validation by MRM- based assays was carried out for three proteins identified from discovery studies.

    Journal: Clinical proteomics

    Article Title: Proteomic analysis of human osteoarthritis synovial fluid

    doi: 10.1186/1559-0275-11-6

    Figure Lengend Snippet: Work flow illustrating the steps involved in the proteomic analysis of OA synovial fluid. OA synovial fluid samples were pooled and subjected to depletion of abundant proteins by MARS-6 LC column and lectin affinity chromatography using three different lectins (Concanavalin A, wheat germ agglutinin and jacalin). The depleted fraction was then subjected to SDS-PAGE, SCX and OFFGEL fractionation. The lectin enriched fraction was subjected to SDS-PAGE analysis and SCX fractionation. All fractions were analyzed on LTQ-Orbitrap Velos mass spectrometer. Sequest and Mascot algorithms were used to perform database searches. Subsequently, gene ontology-based functional characterization of the identified synovial fluid proteins was carried out. Further, validation by MRM- based assays was carried out for three proteins identified from discovery studies.

    Article Snippet: For glycoprotein enrichment, the samples were incubated with a mixture of three agarose conjugated lectins- concanavalin A (Con A), wheat germ agglutinin and jacalin (Vector labs, USA) for 12 h at 4˚C.

    Techniques: Flow Cytometry, Affinity Chromatography, SDS Page, Fractionation, Mass Spectrometry, Functional Assay

    N -Glycosylated proteins with high-mannose oligosaccharide chains are much less abundant in the tonoplast than in the total membrane fraction. Microsomal fractions (M) from Arabidopsis protoplasts or purified vacuoles were analyzed by SDS–PAGE and protein blot followed by incubation with concanavalin A conjugated to peroxidase. Numbers on the left indicate the positions of molecular mass markers, in kDa.

    Journal: Journal of Experimental Botany

    Article Title: The Arabidopsis tonoplast is almost devoid of glycoproteins with complex N-glycans, unlike the rat lysosomal membrane

    doi: 10.1093/jxb/erv567

    Figure Lengend Snippet: N -Glycosylated proteins with high-mannose oligosaccharide chains are much less abundant in the tonoplast than in the total membrane fraction. Microsomal fractions (M) from Arabidopsis protoplasts or purified vacuoles were analyzed by SDS–PAGE and protein blot followed by incubation with concanavalin A conjugated to peroxidase. Numbers on the left indicate the positions of molecular mass markers, in kDa.

    Article Snippet: An equal volume, or equal amount of proteins, of soluble or membrane fractions was analyzed by SDS–PAGE and protein blot on a nitrocellulose membrane (Perkin-Elmer), and incubated with appropriate antibodies and anti-rabbit or anti-chicken IgG–peroxidase conjugate (Pierce) For detection of proteins with high-mannose N -glycans, the protein blot was incubated with 3 µg ml–1 concanavalin A (ConA)–peroxidase conjugate (Sigma-Aldrich St. Louis, MO, USA) in phosphate-buffered saline (PBS) containing 0.05% (v/v) Tween-20, 1mM CaCl2 , 1mM MnCl2 , and 1mM MgCl2 for 16h at 20 °C, according to the manufacturer’s protocols.

    Techniques: Purification, SDS Page, Incubation

    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

    Induction of IFNγ-producing antigen specific CD8 +  T cells following i.d. administrations of WT1(peptide)-PSNPs candidates in HLA-A2.1/Kb mice. WT1 derived peptides (WT1A, WT1B, WT1C, WT1D, and WT1E) were covalently conjugated to PSNPs to constitute PSNPs vaccine formulations (containing 0.5 mg/ml of each peptide in each of the conjugation mix). Mice were immunized 3 times with each formulation (100 μl or 50 μg (including both conjugated and non-conjugated peptide)/injection) intradermally, 10 days apart. 11 days after the last immunization, antigen specific T cell responses were evaluated by IFN γ ELISpot assay upon stimulations with WT1 peptides (5 μg/ml) or controls (media alone or Con A). Each condition was tested in triplicate on splenocytes from individual mouse ( n  = 4). Results are expressed as stimulation index (SI) of the SFU over the background (media alone) ± SD ( n  = 4 individual mice). Two-way ANOVA analysis indicated the significance of WT1A and WT1B peptide processing in the WT1peptide-PSNPs formulations.  * p

    Journal: Frontiers in Immunology

    Article Title: Design of Peptide-Based Nanovaccines Targeting Leading Antigens From Gynecological Cancers to Induce HLA-A2.1 Restricted CD8+ T Cell Responses

    doi: 10.3389/fimmu.2018.02968

    Figure Lengend Snippet: Induction of IFNγ-producing antigen specific CD8 + T cells following i.d. administrations of WT1(peptide)-PSNPs candidates in HLA-A2.1/Kb mice. WT1 derived peptides (WT1A, WT1B, WT1C, WT1D, and WT1E) were covalently conjugated to PSNPs to constitute PSNPs vaccine formulations (containing 0.5 mg/ml of each peptide in each of the conjugation mix). Mice were immunized 3 times with each formulation (100 μl or 50 μg (including both conjugated and non-conjugated peptide)/injection) intradermally, 10 days apart. 11 days after the last immunization, antigen specific T cell responses were evaluated by IFN γ ELISpot assay upon stimulations with WT1 peptides (5 μg/ml) or controls (media alone or Con A). Each condition was tested in triplicate on splenocytes from individual mouse ( n = 4). Results are expressed as stimulation index (SI) of the SFU over the background (media alone) ± SD ( n = 4 individual mice). Two-way ANOVA analysis indicated the significance of WT1A and WT1B peptide processing in the WT1peptide-PSNPs formulations. * p

    Article Snippet: Splenocytes (50 μl) from immunized mice (2 × 107 cells/ml, either individual or pooled) were added to triplicate wells and incubated with 50 μl of recall antigens (see figure legends for specific details for respective experiment) at various concentrations (2.5–25 μg/ml final for all potential CD8+ epitopes and 25–100 μg/ml final for long peptides and protein) at 37°C incubator filled with 5% CO2 for a minimum of 16 h. Concanavalin A (Con-A) (1 μg/ml final, Amersham Biosciences, Uppsala, Sweden) was used as a positive control and background wells were added with CM only.

    Techniques: Mouse Assay, Derivative Assay, Conjugation Assay, Injection, Enzyme-linked Immunospot

    Antigen-specific T cell responses in HLA-A2.1/Kb mice induced by SV peptides with CpG or PSNPs. SV-derived peptides:  (A)  SV01 and SV02,  (B)  SV10,  (C)  SV10, SV12, SV13, SV14, and SV16 were covalently conjugated to PSNPs forming PSNPs vaccine formulations. Each formulation contained equal amount of each SV peptide target and PSNPs (all at 0.5 mg/ml per peptide, 1% solid for PSNPs; 100 μl/injection). Equivalent amount of SV01, SV02, and SV10 peptides were also mixed with CpG (20 μg/injection) as comparison. For each immunization group, mice were immunized 3 times intradermally, 10 days apart. 11 days after the last immunization, antigen specific T cell responses were evaluated by IFN-γ ELISpot assay upon stimulations with antigen specific peptides (dosages on the figure (μg/ml) except C all at 25 μg/ml) or controls (media alone, or Con A). Each condition was tested in triplicate on splenocytes from individual mouse ( n  = 3–4). Results are expressed as the Stimulation Index (SI) of the antigen-induced IFN-γ responses (measured by SFU) over the background levels (media alone responses) ± SD ( n  = 4 individual mice) upon stimulation for each peptide conditions assayed in triplicated wells. Two-way ANOVA analysis indicated the significance of antigen specific responses induced by specific peptides in the SVpeptide-PSNPs or SVpeptide+CpG formulations.  * p

    Journal: Frontiers in Immunology

    Article Title: Design of Peptide-Based Nanovaccines Targeting Leading Antigens From Gynecological Cancers to Induce HLA-A2.1 Restricted CD8+ T Cell Responses

    doi: 10.3389/fimmu.2018.02968

    Figure Lengend Snippet: Antigen-specific T cell responses in HLA-A2.1/Kb mice induced by SV peptides with CpG or PSNPs. SV-derived peptides: (A) SV01 and SV02, (B) SV10, (C) SV10, SV12, SV13, SV14, and SV16 were covalently conjugated to PSNPs forming PSNPs vaccine formulations. Each formulation contained equal amount of each SV peptide target and PSNPs (all at 0.5 mg/ml per peptide, 1% solid for PSNPs; 100 μl/injection). Equivalent amount of SV01, SV02, and SV10 peptides were also mixed with CpG (20 μg/injection) as comparison. For each immunization group, mice were immunized 3 times intradermally, 10 days apart. 11 days after the last immunization, antigen specific T cell responses were evaluated by IFN-γ ELISpot assay upon stimulations with antigen specific peptides (dosages on the figure (μg/ml) except C all at 25 μg/ml) or controls (media alone, or Con A). Each condition was tested in triplicate on splenocytes from individual mouse ( n = 3–4). Results are expressed as the Stimulation Index (SI) of the antigen-induced IFN-γ responses (measured by SFU) over the background levels (media alone responses) ± SD ( n = 4 individual mice) upon stimulation for each peptide conditions assayed in triplicated wells. Two-way ANOVA analysis indicated the significance of antigen specific responses induced by specific peptides in the SVpeptide-PSNPs or SVpeptide+CpG formulations. * p

    Article Snippet: Splenocytes (50 μl) from immunized mice (2 × 107 cells/ml, either individual or pooled) were added to triplicate wells and incubated with 50 μl of recall antigens (see figure legends for specific details for respective experiment) at various concentrations (2.5–25 μg/ml final for all potential CD8+ epitopes and 25–100 μg/ml final for long peptides and protein) at 37°C incubator filled with 5% CO2 for a minimum of 16 h. Concanavalin A (Con-A) (1 μg/ml final, Amersham Biosciences, Uppsala, Sweden) was used as a positive control and background wells were added with CM only.

    Techniques: Mouse Assay, Derivative Assay, Injection, Enzyme-linked Immunospot

    Antigen-specific T cell responses in HLA-A2.1/Kb mice induced by HPV peptides with CpG or PSNPs. HPV01, -HPV05 and –HPV08 peptides were either mixed with CpG or covalently conjugated to PSNPs forming nanovaccine formulations. Each formulation was injected with matching amount of target peptide antigen (all contained 0.5 μg/peptide antigen/injection in 100–200 μl volume). Matching amount of HPV01, HPV05 and HPV08 peptides were also mixed with CpG (20 μg/injection) as comparison. Mice were immunized once intradermally. 15 days after the immunization, antigen specific T cell responses were evaluated by IFN-γ ELISpot assay upon stimulations with different concentration of antigen specific peptides (5, 10, 20, and 50 μg/ml) or controls (media alone, or Con A). Each condition was tested in triplicate on splenocytes from pooled cells within each group of mice ( n  = 3). Results were expressed as Stimulation Index (SI) of the antigen-induced IFN-γ responses (measured by SFU) over the background levels (media alone responses) (± SD triplicated in assay) upon stimulation with HPV05, HPV08 and HPV01 peptide at 20 μg/ml.  *** p

    Journal: Frontiers in Immunology

    Article Title: Design of Peptide-Based Nanovaccines Targeting Leading Antigens From Gynecological Cancers to Induce HLA-A2.1 Restricted CD8+ T Cell Responses

    doi: 10.3389/fimmu.2018.02968

    Figure Lengend Snippet: Antigen-specific T cell responses in HLA-A2.1/Kb mice induced by HPV peptides with CpG or PSNPs. HPV01, -HPV05 and –HPV08 peptides were either mixed with CpG or covalently conjugated to PSNPs forming nanovaccine formulations. Each formulation was injected with matching amount of target peptide antigen (all contained 0.5 μg/peptide antigen/injection in 100–200 μl volume). Matching amount of HPV01, HPV05 and HPV08 peptides were also mixed with CpG (20 μg/injection) as comparison. Mice were immunized once intradermally. 15 days after the immunization, antigen specific T cell responses were evaluated by IFN-γ ELISpot assay upon stimulations with different concentration of antigen specific peptides (5, 10, 20, and 50 μg/ml) or controls (media alone, or Con A). Each condition was tested in triplicate on splenocytes from pooled cells within each group of mice ( n = 3). Results were expressed as Stimulation Index (SI) of the antigen-induced IFN-γ responses (measured by SFU) over the background levels (media alone responses) (± SD triplicated in assay) upon stimulation with HPV05, HPV08 and HPV01 peptide at 20 μg/ml. *** p

    Article Snippet: Splenocytes (50 μl) from immunized mice (2 × 107 cells/ml, either individual or pooled) were added to triplicate wells and incubated with 50 μl of recall antigens (see figure legends for specific details for respective experiment) at various concentrations (2.5–25 μg/ml final for all potential CD8+ epitopes and 25–100 μg/ml final for long peptides and protein) at 37°C incubator filled with 5% CO2 for a minimum of 16 h. Concanavalin A (Con-A) (1 μg/ml final, Amersham Biosciences, Uppsala, Sweden) was used as a positive control and background wells were added with CM only.

    Techniques: Mouse Assay, Injection, Enzyme-linked Immunospot, Concentration Assay

    Impact of immunization schedules and time interval on HPV08-PSNPs immunogenicity. HPV08 peptides were covalently conjugated to PSNPs forming HPV08-PSNPs nanovaccine formulation (final containing 0.37 mg/ml of HPV08 conjugated to PSNPs, 100 μl (or 37 μg)/injection). Mice were immunized following the schedules listed in the figure. Twelve days after the last immunization, antigen specific T cell responses were evaluated by IFN-γ ELISpot assay upon stimulations with antigen specific peptides (HPV05 and HPV08, all at 25 μg/ml) or controls (media alone, or Con A). Each condition was tested in triplicate on splenocytes from individual mouse ( n  = 4). Results are expressed as net spot-forming-unit (SFU)/million splenocytes/mouse upon each peptide recall ± SD ( n  = 4 individual mice). Two-way ANOVA analysis indicated the significance of HPV05 and HPV08 peptides induced specific responses in the HPV08-PSNPs formulations  * p

    Journal: Frontiers in Immunology

    Article Title: Design of Peptide-Based Nanovaccines Targeting Leading Antigens From Gynecological Cancers to Induce HLA-A2.1 Restricted CD8+ T Cell Responses

    doi: 10.3389/fimmu.2018.02968

    Figure Lengend Snippet: Impact of immunization schedules and time interval on HPV08-PSNPs immunogenicity. HPV08 peptides were covalently conjugated to PSNPs forming HPV08-PSNPs nanovaccine formulation (final containing 0.37 mg/ml of HPV08 conjugated to PSNPs, 100 μl (or 37 μg)/injection). Mice were immunized following the schedules listed in the figure. Twelve days after the last immunization, antigen specific T cell responses were evaluated by IFN-γ ELISpot assay upon stimulations with antigen specific peptides (HPV05 and HPV08, all at 25 μg/ml) or controls (media alone, or Con A). Each condition was tested in triplicate on splenocytes from individual mouse ( n = 4). Results are expressed as net spot-forming-unit (SFU)/million splenocytes/mouse upon each peptide recall ± SD ( n = 4 individual mice). Two-way ANOVA analysis indicated the significance of HPV05 and HPV08 peptides induced specific responses in the HPV08-PSNPs formulations * p

    Article Snippet: Splenocytes (50 μl) from immunized mice (2 × 107 cells/ml, either individual or pooled) were added to triplicate wells and incubated with 50 μl of recall antigens (see figure legends for specific details for respective experiment) at various concentrations (2.5–25 μg/ml final for all potential CD8+ epitopes and 25–100 μg/ml final for long peptides and protein) at 37°C incubator filled with 5% CO2 for a minimum of 16 h. Concanavalin A (Con-A) (1 μg/ml final, Amersham Biosciences, Uppsala, Sweden) was used as a positive control and background wells were added with CM only.

    Techniques: Injection, Mouse Assay, Enzyme-linked Immunospot

    Imaging of purified tissue cysts A–C Assembly of the Cytospin holders to pellet the purified tissue cysts onto the slide surface. A . A glass slide and hole punched filter card are placed into the stainless steel clip holder. B . A sample funnel is placed into the holder such that the base of funnel aligns with the lower hole punch on the filter card. C . Assembled Cytospin holders. D–E . Cytospin holders are placed in the centrifuge and the sample added to the funnels using a micropipettor. F . Following centrifugation and the disassembly of the holder the slides are placed in a Coplin jar with fixative. G–I Imaging of a tissue cyst with Dolichos biflorus lectin (DBA-green) ( G ) and Concanavalin A (ConA-Red) ( H ) acquired on a Zeiss Axiophot stand using a 100× 1.4NA objective. The merged image ( I ) clearly shows the differential distribution of the glycans recognized by these lectins within the tissue cyst.

    Journal: Current protocols in microbiology

    Article Title: Toxoplasma gondii tissue cyst purification using Percoll gradients

    doi: 10.1002/cpmc.30

    Figure Lengend Snippet: Imaging of purified tissue cysts A–C Assembly of the Cytospin holders to pellet the purified tissue cysts onto the slide surface. A . A glass slide and hole punched filter card are placed into the stainless steel clip holder. B . A sample funnel is placed into the holder such that the base of funnel aligns with the lower hole punch on the filter card. C . Assembled Cytospin holders. D–E . Cytospin holders are placed in the centrifuge and the sample added to the funnels using a micropipettor. F . Following centrifugation and the disassembly of the holder the slides are placed in a Coplin jar with fixative. G–I Imaging of a tissue cyst with Dolichos biflorus lectin (DBA-green) ( G ) and Concanavalin A (ConA-Red) ( H ) acquired on a Zeiss Axiophot stand using a 100× 1.4NA objective. The merged image ( I ) clearly shows the differential distribution of the glycans recognized by these lectins within the tissue cyst.

    Article Snippet: Shandon Cytospin 4 Centrifuge Cytospin funnels (TPX Sample Chamber) (Thermo #A78710018) Cytoclip stainless steel slide holders (Thermo Fisher #59-910-052) Cytospin filter cards (Fisher #22-030-410) Frosted Glass Slides (Fisher #12-544-2) Cardboard slide holder (Fisher #12-587-10) Wax pencil (Fisher #NC9072020) Copin jars with screw on cap (Fisher 08-816) 70% Ethanol for decontamination Absolute Methanol (at −20°C) Paraformaldehyde stock (EMS #RT15710) Triton X-100 (0.2% in PBS) (Sigma #X100) Carbo-Free Blocking Solution (Vector Laboratories #SP5040) Dolichos biflorus Lectin (FITC-conjugated) (Vector Laboratories #FL-1031) Concanavalin A Lectin (Rhodamine conjugated) Vector Laboratories #FL-1001) Hoescht Dye (10mg/ml) (ThermoFisher #H3569)

    Techniques: Imaging, Purification, Cross-linking Immunoprecipitation, Transferring, Centrifugation

    Sunitinib MP reduce leukostasis and improve retinal perfusion in rho/VEGF mice. Rho/VEGF  mice were given an intravitreous injection of 10 µg of Suni MP or 40 µg of aflibercept in one eye and 10 µg of Empty MP or PBS in the other eye. After 1 week, mice were perfused through the left ventricle with PBS to remove erythrocytes and leukocytes and then perfused with FITC-concanavalin A. Examination of retinal flat mounts by fluorescence microscopy showed a significant reduction in mean number of adherent intravascular leukocytes per retina in Suni MP-injected eyes compared with Empty MP-injected fellow eyes and in aflibercept-injected eyes compared with PBS-injected fellow eyes ( a  scale bar = 100 µm; * p

    Journal: Nature Communications

    Article Title: Sustained treatment of retinal vascular diseases with self-aggregating sunitinib microparticles

    doi: 10.1038/s41467-020-14340-x

    Figure Lengend Snippet: Sunitinib MP reduce leukostasis and improve retinal perfusion in rho/VEGF mice. Rho/VEGF mice were given an intravitreous injection of 10 µg of Suni MP or 40 µg of aflibercept in one eye and 10 µg of Empty MP or PBS in the other eye. After 1 week, mice were perfused through the left ventricle with PBS to remove erythrocytes and leukocytes and then perfused with FITC-concanavalin A. Examination of retinal flat mounts by fluorescence microscopy showed a significant reduction in mean number of adherent intravascular leukocytes per retina in Suni MP-injected eyes compared with Empty MP-injected fellow eyes and in aflibercept-injected eyes compared with PBS-injected fellow eyes ( a scale bar = 100 µm; * p

    Article Snippet: Mice were then perfused over a span of 2–3 min with rhodamine- or FITC-labeled conA (20 µg/ml in PBS, 5 mg/kg; RL-1002 and FL-1001; Vector Labs Thermofisher).

    Techniques: Mouse Assay, Injection, Fluorescence, Microscopy