nunc maxisorptm 96 well plates  (Thermo Fisher)


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    Structured Review

    Thermo Fisher nunc maxisorptm 96 well plates
    Nunc Maxisorptm 96 Well Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nunc maxisorptm 96 well plates/product/Thermo Fisher
    Average 83 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nunc maxisorptm 96 well plates - by Bioz Stars, 2020-03
    83/100 stars

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    Recombinant:

    Article Title: Characterization of a vraG Mutant in a Genetically Stable Staphylococcus aureus Small-Colony Variant and Preliminary Assessment for Use as a Live-Attenuated Vaccine against Intrammamary Infections
    Article Snippet: .. For target antigens, Nunc MaxiSorpTM 96-well plates (Thermo Fisher Scientific Inc., Rochester, NY) were coated with 100 μl of each of the whole S . aureus cell extracts or of the recombinant IsdH protein (10 μg/ml diluted in carbonate/bicarbonate buffer, Sigma), and incubated overnight at room temperature. .. The plates were then saturated with PBS containing 5% skim milk powder for 1 h at 37°C, followed by a second blocking step with an addition of 5% porcine serum to prevent unspecific S . aureus protein A interactions, in the case of whole-cell extracts.

    Incubation:

    Article Title: Vaccination with a live-attenuated small-colony variant improves the humoral and cell-mediated responses against Staphylococcus aureus
    Article Snippet: .. Briefly, Nunc MaxiSorpTM 96-well plates (Thermo Fisher Scientific) were coated with 100 μl of whole S . aureus cell extract (10 μg/ml diluted in carbonate/bicarbonate buffer, Sigma) and incubated overnight at room temperature. .. The plates were then saturated with PBS containing 5% skim milk for 1 h at 37°C, followed by a second blocking step with the addition of 5% porcine serum to prevent unspecific interactions with S . aureus protein A and other staphylococcal immunoglobulin binding proteins [ ].

    Article Title: Characterization of a vraG Mutant in a Genetically Stable Staphylococcus aureus Small-Colony Variant and Preliminary Assessment for Use as a Live-Attenuated Vaccine against Intrammamary Infections
    Article Snippet: .. For target antigens, Nunc MaxiSorpTM 96-well plates (Thermo Fisher Scientific Inc., Rochester, NY) were coated with 100 μl of each of the whole S . aureus cell extracts or of the recombinant IsdH protein (10 μg/ml diluted in carbonate/bicarbonate buffer, Sigma), and incubated overnight at room temperature. .. The plates were then saturated with PBS containing 5% skim milk powder for 1 h at 37°C, followed by a second blocking step with an addition of 5% porcine serum to prevent unspecific S . aureus protein A interactions, in the case of whole-cell extracts.

    Blocking Assay:

    Article Title: Vaccination with a live-attenuated small-colony variant improves the humoral and cell-mediated responses against Staphylococcus aureus
    Article Snippet: Briefly, Nunc MaxiSorpTM 96-well plates (Thermo Fisher Scientific) were coated with 100 μl of whole S . aureus cell extract (10 μg/ml diluted in carbonate/bicarbonate buffer, Sigma) and incubated overnight at room temperature. .. The plates were then saturated with PBS containing 5% skim milk for 1 h at 37°C, followed by a second blocking step with the addition of 5% porcine serum to prevent unspecific interactions with S . aureus protein A and other staphylococcal immunoglobulin binding proteins [ ].

    Article Title: Characterization of a vraG Mutant in a Genetically Stable Staphylococcus aureus Small-Colony Variant and Preliminary Assessment for Use as a Live-Attenuated Vaccine against Intrammamary Infections
    Article Snippet: For target antigens, Nunc MaxiSorpTM 96-well plates (Thermo Fisher Scientific Inc., Rochester, NY) were coated with 100 μl of each of the whole S . aureus cell extracts or of the recombinant IsdH protein (10 μg/ml diluted in carbonate/bicarbonate buffer, Sigma), and incubated overnight at room temperature. .. The plates were then saturated with PBS containing 5% skim milk powder for 1 h at 37°C, followed by a second blocking step with an addition of 5% porcine serum to prevent unspecific S . aureus protein A interactions, in the case of whole-cell extracts.

    Mouse Assay:

    Article Title: Characterization of a vraG Mutant in a Genetically Stable Staphylococcus aureus Small-Colony Variant and Preliminary Assessment for Use as a Live-Attenuated Vaccine against Intrammamary Infections
    Article Snippet: Detection of mouse IgG by ELISA Detection of serum total IgG against the ΔvraG ΔhemB vaccination strain and each of the bovine IMI isolates was performed to demonstrate and measure the systemic humoral response generated by the immunization of mice. .. For target antigens, Nunc MaxiSorpTM 96-well plates (Thermo Fisher Scientific Inc., Rochester, NY) were coated with 100 μl of each of the whole S . aureus cell extracts or of the recombinant IsdH protein (10 μg/ml diluted in carbonate/bicarbonate buffer, Sigma), and incubated overnight at room temperature.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Vaccination with a live-attenuated small-colony variant improves the humoral and cell-mediated responses against Staphylococcus aureus
    Article Snippet: ELISAs Serum total IgG and IgG1/IgG2a isotypes were detected by ELISA against the S . aureus ΔvraG ΔhemB whole cell extract to compare the systemic humoral response generated by the different vaccine versions as previously described [ ]. .. Briefly, Nunc MaxiSorpTM 96-well plates (Thermo Fisher Scientific) were coated with 100 μl of whole S . aureus cell extract (10 μg/ml diluted in carbonate/bicarbonate buffer, Sigma) and incubated overnight at room temperature.

    Article Title: Characterization of a vraG Mutant in a Genetically Stable Staphylococcus aureus Small-Colony Variant and Preliminary Assessment for Use as a Live-Attenuated Vaccine against Intrammamary Infections
    Article Snippet: Paragraph title: Detection of mouse IgG by ELISA ... For target antigens, Nunc MaxiSorpTM 96-well plates (Thermo Fisher Scientific Inc., Rochester, NY) were coated with 100 μl of each of the whole S . aureus cell extracts or of the recombinant IsdH protein (10 μg/ml diluted in carbonate/bicarbonate buffer, Sigma), and incubated overnight at room temperature.

    Generated:

    Article Title: Vaccination with a live-attenuated small-colony variant improves the humoral and cell-mediated responses against Staphylococcus aureus
    Article Snippet: ELISAs Serum total IgG and IgG1/IgG2a isotypes were detected by ELISA against the S . aureus ΔvraG ΔhemB whole cell extract to compare the systemic humoral response generated by the different vaccine versions as previously described [ ]. .. Briefly, Nunc MaxiSorpTM 96-well plates (Thermo Fisher Scientific) were coated with 100 μl of whole S . aureus cell extract (10 μg/ml diluted in carbonate/bicarbonate buffer, Sigma) and incubated overnight at room temperature.

    Article Title: Characterization of a vraG Mutant in a Genetically Stable Staphylococcus aureus Small-Colony Variant and Preliminary Assessment for Use as a Live-Attenuated Vaccine against Intrammamary Infections
    Article Snippet: Detection of mouse IgG by ELISA Detection of serum total IgG against the ΔvraG ΔhemB vaccination strain and each of the bovine IMI isolates was performed to demonstrate and measure the systemic humoral response generated by the immunization of mice. .. For target antigens, Nunc MaxiSorpTM 96-well plates (Thermo Fisher Scientific Inc., Rochester, NY) were coated with 100 μl of each of the whole S . aureus cell extracts or of the recombinant IsdH protein (10 μg/ml diluted in carbonate/bicarbonate buffer, Sigma), and incubated overnight at room temperature.

    Binding Assay:

    Article Title: Vaccination with a live-attenuated small-colony variant improves the humoral and cell-mediated responses against Staphylococcus aureus
    Article Snippet: Briefly, Nunc MaxiSorpTM 96-well plates (Thermo Fisher Scientific) were coated with 100 μl of whole S . aureus cell extract (10 μg/ml diluted in carbonate/bicarbonate buffer, Sigma) and incubated overnight at room temperature. .. The plates were then saturated with PBS containing 5% skim milk for 1 h at 37°C, followed by a second blocking step with the addition of 5% porcine serum to prevent unspecific interactions with S . aureus protein A and other staphylococcal immunoglobulin binding proteins [ ].

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    Thermo Fisher 96 well elisa plates
    Specificity of <t>ELISA</t> and RT–PCR assays The specificity of the PTX3 ELISA was tested using different dilutions of 2C3 antibody to measure immobilized murine and human PTX3. Purified recombinant murine and human PTX3 were immobilized in <t>96‐well</t> ELISA plates, and then, different dilutions of 2C3 were added. The graph shows dose–response of 2C3 on immobilized murine or human PTX3. Human PTX3 was not detected by 2C3 antibody. Evaluation of the amplification efficiency of real‐time RT–PCR assay designed for PTX3 expression in astrocyte cell cultures. (B, C) Melting curve and amplification plot of PTX3 RT–qPCR assay. (D) Standard curves of PTX3 and GAPDH, used as reference mRNA, obtained using fivefold serial dilutions of the cDNA (420, 84, 16.8, 3.36 ng). The threshold cycle ( C t ) values ( y ‐axis) are plotted against log 10 values of cDNA input amounts ( x ‐axis). The graphs are parallel lines and the calculated efficiencies (E) are, respectively, of 1.13 and 1.12 from a y‐slope of −3.04 and −3.07 and a correlation coefficient ( R 2 ) > 0.9.
    96 Well Elisa Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/96 well elisa plates/product/Thermo Fisher
    Average 99 stars, based on 104 article reviews
    Price from $9.99 to $1999.99
    96 well elisa plates - by Bioz Stars, 2020-03
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    99
    Thermo Fisher elisa plates
    Description and characterization of the chimeric human <t>FasL-derived</t> constructs Panel A: Schematic representation of soluble FasL (sFasL), Flag-tagged sFasL (sfFasL), polymeric Flag-tagged soluble FasL (pfFasL), polymeric TCR γ4 and δ5 Flag-tagged soluble FasL generating the TCR-pfFasL upon cotransfection, and beta2-microglobulin-fused HLA-A*02: 01 Flag-tagged soluble FasL (HLA-pfFasL). The f and p symbols represent the flag epitope and the LIF receptor-derived domain triggering the polymerisation of the FasL oligomers, respectively. Panel B: direct immunoblot of the supernatants from COS cells transfected with the empty vector (control) or the FasL constructs sFasL, sfFasL and pfFasL. Panel C: immunoprecipitation of the TCR-pfFasL chimera from transfected HEK cells, using an irrelevant IgG1 antibody, the anti-Flag (clone M2), the anti-FasL (clone 10F2), the anti-TCRγδ (clone IMU-510) or the anti-TCRδ5 (clone 12C7) antibodies. Panel D: immunoprecipitation of the HLA-pfFasL chimera from the supernatant of COS cells, with anti-Flag, anti-FasL or anti-β2microglobulin antibodies. As controls, the same experiment was performed with irrelevant IgG1 and IgG2 antibodies. Panel E: cytotoxic effect of the FasL chimeras. The indicated chimeras, as supernatants from transfected cells and quantitated using the <t>ELISA</t> for FasL, were incubated at the indicated concentrations with Jurkat cells. After 18 h, the MTT cell viability assay was performed. The anti-Flag M2 antibody at 0.5 µg/ml was added to sfFasL to render it cytotoxic.
    Elisa Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 430 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/elisa plates/product/Thermo Fisher
    Average 99 stars, based on 430 article reviews
    Price from $9.99 to $1999.99
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    78
    Thermo Fisher grp78 elisa microtiter 96 well plates
    Analysis of <t>GRP78–HTJ1</t> and OxPL–GRP78 interactions. (A) GRP78 and HTJ1 expression in HPAEC and human lung microvascular endothelial cells was detected by Western Blot. (B, C) GRP78 interactions were analyzed in coimmunoprecipitation assays using lysates from control or DMPC- (10 μg/ml, 15 min) or OxPAPC-stimulated (10 μg/ml) cells with antibody to GRP78 (B, top), HTJ1 (B, bottom), or EO6 antibody recognizing OxPL (C). (D) Human recombinant GRP78 was incubated with OxPAPC, OxPAPS, or their oxidation-resistant analogues DMPC or DMPS. Left, native gel electrophoresis, followed by Western blot with anti-GRP78 antibody. Shift in electrophoretic mobility of GRP78 incubated with OxPAPS, but not DMPS, indicates formation of GRP78–OxPAPS complex. Right, SDS–PAGE, followed by Western blot with EO6 antibody and reprobing with anti-GRP78 antibody. Positive EO6 immunoreactivity of GRP78 preincubated with OxPAPC indicates formation of GRP78–OxPAPC complex. (E) <t>ELISA</t> plates coated with OxPAPS or DMPS or control uncoated plates incubated with PBS incubated with human recombinant GRP78 (left) or HPAEC lysates (middle and right). The bound GRP78 was detected using anti-GRP78 antibody. * p
    Grp78 Elisa Microtiter 96 Well Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    grp78 elisa microtiter 96 well plates - by Bioz Stars, 2020-03
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    Specificity of ELISA and RT–PCR assays The specificity of the PTX3 ELISA was tested using different dilutions of 2C3 antibody to measure immobilized murine and human PTX3. Purified recombinant murine and human PTX3 were immobilized in 96‐well ELISA plates, and then, different dilutions of 2C3 were added. The graph shows dose–response of 2C3 on immobilized murine or human PTX3. Human PTX3 was not detected by 2C3 antibody. Evaluation of the amplification efficiency of real‐time RT–PCR assay designed for PTX3 expression in astrocyte cell cultures. (B, C) Melting curve and amplification plot of PTX3 RT–qPCR assay. (D) Standard curves of PTX3 and GAPDH, used as reference mRNA, obtained using fivefold serial dilutions of the cDNA (420, 84, 16.8, 3.36 ng). The threshold cycle ( C t ) values ( y ‐axis) are plotted against log 10 values of cDNA input amounts ( x ‐axis). The graphs are parallel lines and the calculated efficiencies (E) are, respectively, of 1.13 and 1.12 from a y‐slope of −3.04 and −3.07 and a correlation coefficient ( R 2 ) > 0.9.

    Journal: The EMBO Journal

    Article Title: Pentraxin 3 regulates synaptic function by inducing AMPA receptor clustering via ECM remodeling and β1‐integrin

    doi: 10.15252/embj.201899529

    Figure Lengend Snippet: Specificity of ELISA and RT–PCR assays The specificity of the PTX3 ELISA was tested using different dilutions of 2C3 antibody to measure immobilized murine and human PTX3. Purified recombinant murine and human PTX3 were immobilized in 96‐well ELISA plates, and then, different dilutions of 2C3 were added. The graph shows dose–response of 2C3 on immobilized murine or human PTX3. Human PTX3 was not detected by 2C3 antibody. Evaluation of the amplification efficiency of real‐time RT–PCR assay designed for PTX3 expression in astrocyte cell cultures. (B, C) Melting curve and amplification plot of PTX3 RT–qPCR assay. (D) Standard curves of PTX3 and GAPDH, used as reference mRNA, obtained using fivefold serial dilutions of the cDNA (420, 84, 16.8, 3.36 ng). The threshold cycle ( C t ) values ( y ‐axis) are plotted against log 10 values of cDNA input amounts ( x ‐axis). The graphs are parallel lines and the calculated efficiencies (E) are, respectively, of 1.13 and 1.12 from a y‐slope of −3.04 and −3.07 and a correlation coefficient ( R 2 ) > 0.9.

    Article Snippet: Briefly, 96‐well ELISA plates (Nunc MaxiSorp, Thermo Fischer Scientific, Roskilde, Denmark) were coated with monoclonal antibody 2C3 anti‐mouse PTX3 in coating buffer (15 mM carbonate buffer pH 9.6) and incubated overnight at 4°C.

    Techniques: Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Purification, Recombinant, Amplification, Quantitative RT-PCR, Expressing

    Natively purified Hic construct interact with hTSP-1. A , Hic peptide selection dependent on putative factor H-binding regions. Shown is the amino acid sequence of recombinant Hic2 (without His 6 tag). Putative factor H-binding regions are highlighted in red , based on the publication of Jarva et al. ). Recombinant Hic5/6/7 peptides are indicated with blue lines. B , secondary structure prediction for Hic. Secondary structure prediction for the recombinant Hic construct Hic2 ( S. pneumoniae A66, residues 38–245) using Netsurfp and Jpret4. For Netsurfp a 50% threshold was set to determine the probability for α-helical regions. Matching predictions of both programs are shown as α-helices ( yellow ). C , schematic model of Hic of S. pneumoniae serotype 3 (A66) and heterologously expressed His 6 -tagged fragments. LP , leader peptide; P , proline-rich sequence; LPSTG , sortase anchoring motif. D , SDS-PAGE of heterologously expressed Hic fragments (Hic2, Hic5, Hic6, and Hic7) stained with Coomassie Brilliant Blue R250. Lane M , Fermentas-prestained protein ladder. E , dose-dependent binding of soluble pneumococcal Hic to immobilized hTSP-1 and human factor H (positive control). Human TSP-1 and factor H were coated on microtiter plates and, after blocking, incubated with increasing molecular ratios of recombinant Hic-fragments. Bound pneumococcal proteins were detected using a polyclonal mouse anti-Hic antibody and a peroxidase-coupled secondary anti-mouse antibody. F , concentration-dependent binding of soluble hTSP-1 to immobilized, heterologously expressed Hic proteins and PspC3 SH13 (positive control). Proteins were immobilized on microtiter plates in equimolar amounts related to Hic7 (0.5 μg in 100 μl/cavity). Binding of hTSP-1 was detected, after incubating the immobilized proteins with increasing concentrations of hTSP-1 (0–25 μg/ml), using a specific polyclonal mouse anti-hTSP-1 antibody and a peroxidase-coupled secondary anti-mouse antibody. In E and F , results are illustrated as mean values ± S.D. of at least three independent experiments. The values of control wells without soluble overlay protein were subtracted from each measured value. ns , not significant; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Serotype 3 pneumococci sequester platelet-derived human thrombospondin-1 via the adhesin and immune evasion protein Hic

    doi: 10.1074/jbc.M116.760504

    Figure Lengend Snippet: Natively purified Hic construct interact with hTSP-1. A , Hic peptide selection dependent on putative factor H-binding regions. Shown is the amino acid sequence of recombinant Hic2 (without His 6 tag). Putative factor H-binding regions are highlighted in red , based on the publication of Jarva et al. ). Recombinant Hic5/6/7 peptides are indicated with blue lines. B , secondary structure prediction for Hic. Secondary structure prediction for the recombinant Hic construct Hic2 ( S. pneumoniae A66, residues 38–245) using Netsurfp and Jpret4. For Netsurfp a 50% threshold was set to determine the probability for α-helical regions. Matching predictions of both programs are shown as α-helices ( yellow ). C , schematic model of Hic of S. pneumoniae serotype 3 (A66) and heterologously expressed His 6 -tagged fragments. LP , leader peptide; P , proline-rich sequence; LPSTG , sortase anchoring motif. D , SDS-PAGE of heterologously expressed Hic fragments (Hic2, Hic5, Hic6, and Hic7) stained with Coomassie Brilliant Blue R250. Lane M , Fermentas-prestained protein ladder. E , dose-dependent binding of soluble pneumococcal Hic to immobilized hTSP-1 and human factor H (positive control). Human TSP-1 and factor H were coated on microtiter plates and, after blocking, incubated with increasing molecular ratios of recombinant Hic-fragments. Bound pneumococcal proteins were detected using a polyclonal mouse anti-Hic antibody and a peroxidase-coupled secondary anti-mouse antibody. F , concentration-dependent binding of soluble hTSP-1 to immobilized, heterologously expressed Hic proteins and PspC3 SH13 (positive control). Proteins were immobilized on microtiter plates in equimolar amounts related to Hic7 (0.5 μg in 100 μl/cavity). Binding of hTSP-1 was detected, after incubating the immobilized proteins with increasing concentrations of hTSP-1 (0–25 μg/ml), using a specific polyclonal mouse anti-hTSP-1 antibody and a peroxidase-coupled secondary anti-mouse antibody. In E and F , results are illustrated as mean values ± S.D. of at least three independent experiments. The values of control wells without soluble overlay protein were subtracted from each measured value. ns , not significant; *, p

    Article Snippet: Cavities of microtiter plates (96-well, MaxisorpTM ; Nunc, Thermo Fisher Scientific) were coated with 0.1 μg/well hTSP-1 or human factor H as control in PBS (pH 7.4) overnight at 4 °C.

    Techniques: Purification, Hydrophobic Interaction Chromatography, Construct, Selection, Binding Assay, Sequencing, Recombinant, SDS Page, Staining, Positive Control, Blocking Assay, Incubation, Concentration Assay

    Human TSP-1 recognizes peptide domains within Hic similar to vitronectin and factor H. A and B , pneumococcal proteins were immobilized on microtiter plates (Polysorp TM ; Nunc) in equimolar amounts related to Hic7 (0.5 μg in 100 μl/well). Binding of a constant concentration of hTSP-1 (5 μg/ml) or vitronectin ( Vn ; 1 μg/ml) to immobilized Hic peptides was measured in the presence of increasing molecular ratios of Vn (related to hTSP-1) or hTSP-1 (related to Vn). Glycoprotein binding was detected either with polyclonal mouse anti-hTSP-1 IgG or polyclonal rabbit anti-human Vn antiserum followed by a secondary HRP-coupled goat anti-mouse IgG or HRP-coupled goat anti-rabbit IgG. C and D , pneumococcal Hic2 construct was immobilized on microtiter plates (Maxisorp TM ; Nunc). Binding of a constant concentration of hTSP-1 (2.5 μg/ml) or factor H ( FH , 1 μg/ml) to immobilized Hic2 was measured in the presence of increasing molecular ratios of factor H (related to hTSP-1) or hTSP-1 (related to factor H) using either a polyclonal mouse anti-hTSP-1 IgG or polyclonal goat anti-human FH IgG followed by a secondary HRP-coupled goat anti-mouse IgG or HRP-coupled rabbit anti-goat IgG. E , after immobilization of Hic2 (Maxisorp TM ; Nunc) binding of increasing molecular ratios of hTSP-1 (related to factor H) was measured in presence of a constant concentration of FH (1 μg/ml). Detection of hTSP-1-binding to factor H and/or Hic2 was analyzed as described in C . In A–E , the results are illustrated as mean values ± S.D. of at least three independent experiments. The values of control wells without soluble overlay protein were subtracted from each measured value. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Serotype 3 pneumococci sequester platelet-derived human thrombospondin-1 via the adhesin and immune evasion protein Hic

    doi: 10.1074/jbc.M116.760504

    Figure Lengend Snippet: Human TSP-1 recognizes peptide domains within Hic similar to vitronectin and factor H. A and B , pneumococcal proteins were immobilized on microtiter plates (Polysorp TM ; Nunc) in equimolar amounts related to Hic7 (0.5 μg in 100 μl/well). Binding of a constant concentration of hTSP-1 (5 μg/ml) or vitronectin ( Vn ; 1 μg/ml) to immobilized Hic peptides was measured in the presence of increasing molecular ratios of Vn (related to hTSP-1) or hTSP-1 (related to Vn). Glycoprotein binding was detected either with polyclonal mouse anti-hTSP-1 IgG or polyclonal rabbit anti-human Vn antiserum followed by a secondary HRP-coupled goat anti-mouse IgG or HRP-coupled goat anti-rabbit IgG. C and D , pneumococcal Hic2 construct was immobilized on microtiter plates (Maxisorp TM ; Nunc). Binding of a constant concentration of hTSP-1 (2.5 μg/ml) or factor H ( FH , 1 μg/ml) to immobilized Hic2 was measured in the presence of increasing molecular ratios of factor H (related to hTSP-1) or hTSP-1 (related to factor H) using either a polyclonal mouse anti-hTSP-1 IgG or polyclonal goat anti-human FH IgG followed by a secondary HRP-coupled goat anti-mouse IgG or HRP-coupled rabbit anti-goat IgG. E , after immobilization of Hic2 (Maxisorp TM ; Nunc) binding of increasing molecular ratios of hTSP-1 (related to factor H) was measured in presence of a constant concentration of FH (1 μg/ml). Detection of hTSP-1-binding to factor H and/or Hic2 was analyzed as described in C . In A–E , the results are illustrated as mean values ± S.D. of at least three independent experiments. The values of control wells without soluble overlay protein were subtracted from each measured value. *, p

    Article Snippet: Cavities of microtiter plates (96-well, MaxisorpTM ; Nunc, Thermo Fisher Scientific) were coated with 0.1 μg/well hTSP-1 or human factor H as control in PBS (pH 7.4) overnight at 4 °C.

    Techniques: Hydrophobic Interaction Chromatography, Binding Assay, Concentration Assay, Construct

    Interaction between Hic2 and BSA. A , microtiter plate (Maxisorp TM ; Nunc) coated with blocking solution containing increasing concentrations of BSA followed by incubation with Hic2. Binding of Hic2 was detected using a polyclonal anti-Hic2 IgG followed by a secondary HRP-coupled goat anti-mouse IgG. The results are illustrated as mean values of one experiment with duplicates. B , interactions of hTSP-1, factor H, and BSA with immobilized Hic2 were analyzed by surface plasmon resonance spectroscopy in manual runs. Therefore, a CM5 sensorchip was coated with native Hic2, and kinetics were conducted in PBS, 0.05% Tween 20 (pH 7.4) with a flow rate of 10 μl/min. The values of the control flow cells were subtracted from each sensorgram. C , binding of Hic2 (10 μg/ml) to immobilized BSA and factor H. Bound Hic was detected using a polyclonal anti-Hic2 IgG and a secondary alkaline phosphatase-coupled goat anti-mouse IgG and NBT/BCIP as substrate.

    Journal: The Journal of Biological Chemistry

    Article Title: Serotype 3 pneumococci sequester platelet-derived human thrombospondin-1 via the adhesin and immune evasion protein Hic

    doi: 10.1074/jbc.M116.760504

    Figure Lengend Snippet: Interaction between Hic2 and BSA. A , microtiter plate (Maxisorp TM ; Nunc) coated with blocking solution containing increasing concentrations of BSA followed by incubation with Hic2. Binding of Hic2 was detected using a polyclonal anti-Hic2 IgG followed by a secondary HRP-coupled goat anti-mouse IgG. The results are illustrated as mean values of one experiment with duplicates. B , interactions of hTSP-1, factor H, and BSA with immobilized Hic2 were analyzed by surface plasmon resonance spectroscopy in manual runs. Therefore, a CM5 sensorchip was coated with native Hic2, and kinetics were conducted in PBS, 0.05% Tween 20 (pH 7.4) with a flow rate of 10 μl/min. The values of the control flow cells were subtracted from each sensorgram. C , binding of Hic2 (10 μg/ml) to immobilized BSA and factor H. Bound Hic was detected using a polyclonal anti-Hic2 IgG and a secondary alkaline phosphatase-coupled goat anti-mouse IgG and NBT/BCIP as substrate.

    Article Snippet: Cavities of microtiter plates (96-well, MaxisorpTM ; Nunc, Thermo Fisher Scientific) were coated with 0.1 μg/well hTSP-1 or human factor H as control in PBS (pH 7.4) overnight at 4 °C.

    Techniques: Blocking Assay, Incubation, Binding Assay, SPR Assay, Spectroscopy, Flow Cytometry, Hydrophobic Interaction Chromatography

    Pneumococcal Hic and PspC target a similar binding domain within hTSP-1, which is different from PavB. Human TSP-1 (0.1 μg in 100 μl/cavity) was immobilized on microtiter plates (Maxisorp TM ; Nunc) and incubated with a constant molecular ratio of recombinant Hic2 fragment (related to hTSP-1) in the presence of increasing molecular ratios of PspC SH13 and PavB SSURE 1–5 (related to Hic2). Bound pneumococcal protein was detected using a polyclonal mouse anti-Hic antibody and a peroxidase-coupled secondary anti-mouse antibody. The mean values of at least three independent experiments are shown with error bars corresponding to S.D. The values of control wells without soluble overlay protein were subtracted from each measured value. ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: Serotype 3 pneumococci sequester platelet-derived human thrombospondin-1 via the adhesin and immune evasion protein Hic

    doi: 10.1074/jbc.M116.760504

    Figure Lengend Snippet: Pneumococcal Hic and PspC target a similar binding domain within hTSP-1, which is different from PavB. Human TSP-1 (0.1 μg in 100 μl/cavity) was immobilized on microtiter plates (Maxisorp TM ; Nunc) and incubated with a constant molecular ratio of recombinant Hic2 fragment (related to hTSP-1) in the presence of increasing molecular ratios of PspC SH13 and PavB SSURE 1–5 (related to Hic2). Bound pneumococcal protein was detected using a polyclonal mouse anti-Hic antibody and a peroxidase-coupled secondary anti-mouse antibody. The mean values of at least three independent experiments are shown with error bars corresponding to S.D. The values of control wells without soluble overlay protein were subtracted from each measured value. ***, p

    Article Snippet: Cavities of microtiter plates (96-well, MaxisorpTM ; Nunc, Thermo Fisher Scientific) were coated with 0.1 μg/well hTSP-1 or human factor H as control in PBS (pH 7.4) overnight at 4 °C.

    Techniques: Hydrophobic Interaction Chromatography, Binding Assay, Incubation, Recombinant

    Description and characterization of the chimeric human FasL-derived constructs Panel A: Schematic representation of soluble FasL (sFasL), Flag-tagged sFasL (sfFasL), polymeric Flag-tagged soluble FasL (pfFasL), polymeric TCR γ4 and δ5 Flag-tagged soluble FasL generating the TCR-pfFasL upon cotransfection, and beta2-microglobulin-fused HLA-A*02: 01 Flag-tagged soluble FasL (HLA-pfFasL). The f and p symbols represent the flag epitope and the LIF receptor-derived domain triggering the polymerisation of the FasL oligomers, respectively. Panel B: direct immunoblot of the supernatants from COS cells transfected with the empty vector (control) or the FasL constructs sFasL, sfFasL and pfFasL. Panel C: immunoprecipitation of the TCR-pfFasL chimera from transfected HEK cells, using an irrelevant IgG1 antibody, the anti-Flag (clone M2), the anti-FasL (clone 10F2), the anti-TCRγδ (clone IMU-510) or the anti-TCRδ5 (clone 12C7) antibodies. Panel D: immunoprecipitation of the HLA-pfFasL chimera from the supernatant of COS cells, with anti-Flag, anti-FasL or anti-β2microglobulin antibodies. As controls, the same experiment was performed with irrelevant IgG1 and IgG2 antibodies. Panel E: cytotoxic effect of the FasL chimeras. The indicated chimeras, as supernatants from transfected cells and quantitated using the ELISA for FasL, were incubated at the indicated concentrations with Jurkat cells. After 18 h, the MTT cell viability assay was performed. The anti-Flag M2 antibody at 0.5 µg/ml was added to sfFasL to render it cytotoxic.

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Description and characterization of the chimeric human FasL-derived constructs Panel A: Schematic representation of soluble FasL (sFasL), Flag-tagged sFasL (sfFasL), polymeric Flag-tagged soluble FasL (pfFasL), polymeric TCR γ4 and δ5 Flag-tagged soluble FasL generating the TCR-pfFasL upon cotransfection, and beta2-microglobulin-fused HLA-A*02: 01 Flag-tagged soluble FasL (HLA-pfFasL). The f and p symbols represent the flag epitope and the LIF receptor-derived domain triggering the polymerisation of the FasL oligomers, respectively. Panel B: direct immunoblot of the supernatants from COS cells transfected with the empty vector (control) or the FasL constructs sFasL, sfFasL and pfFasL. Panel C: immunoprecipitation of the TCR-pfFasL chimera from transfected HEK cells, using an irrelevant IgG1 antibody, the anti-Flag (clone M2), the anti-FasL (clone 10F2), the anti-TCRγδ (clone IMU-510) or the anti-TCRδ5 (clone 12C7) antibodies. Panel D: immunoprecipitation of the HLA-pfFasL chimera from the supernatant of COS cells, with anti-Flag, anti-FasL or anti-β2microglobulin antibodies. As controls, the same experiment was performed with irrelevant IgG1 and IgG2 antibodies. Panel E: cytotoxic effect of the FasL chimeras. The indicated chimeras, as supernatants from transfected cells and quantitated using the ELISA for FasL, were incubated at the indicated concentrations with Jurkat cells. After 18 h, the MTT cell viability assay was performed. The anti-Flag M2 antibody at 0.5 µg/ml was added to sfFasL to render it cytotoxic.

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Derivative Assay, Construct, Cotransfection, FLAG-tag, Transfection, Plasmid Preparation, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Incubation, MTT Assay, Viability Assay

    Direct association of sFasL to the pfFasL-containing chimeric proteins during co-expression. Panel A: Identical amounts of pfFasL (1 µg, according to the Flag ELISA) produced in the presence of the indicated ratios of added sFasL plasmid (left panels) was immunoprecipitated with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (3 µg according to the FasL ELISA, right panel). Panel B: Densitometric detection and quantification of the pfFasL (grey bars) and the sFasL (black bars) fractions, following transfection of the pfFasL plasmid in the presence of the indicated proportion of the sFasL plasmid. The measures were normalized to the condition lacking sFasL. Mean+/- sd from three experiments. Panel C: The TCR-pfFasL chimera (2 µg, according to an ELISA specific for the TCR-pFasL molecule using anti-TCRδ5 (clone 12C7) and anti-FasL (clone 10F2) as capture and tracing antibodies, respectively), produced in the absence or the presence of the sFasL plasmid at the indicated ratio, was immunoprecipitated with the anti-TCRδ5 antibody, then separated by 10% SDS-PAGE under reducing conditions and revealed by immunoblotting with the anti-FasL antibody. As a control, the immunoprecipitation experiment was performed with 2 µg of sFasL protein. Panel D: COS supernatants containing pfFasL (4 µg/ml according to the Flag ELISA) produced alone, was mixed with culture medium or sFasL (15 µg/ml) produced separately in a total volume of 1 ml, and incubated for 24 h at 37°C. Then the recombinant proteins were immunoprecipitated (left panels) with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (15 µg according to the FasL ELISA, right panel).

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Direct association of sFasL to the pfFasL-containing chimeric proteins during co-expression. Panel A: Identical amounts of pfFasL (1 µg, according to the Flag ELISA) produced in the presence of the indicated ratios of added sFasL plasmid (left panels) was immunoprecipitated with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (3 µg according to the FasL ELISA, right panel). Panel B: Densitometric detection and quantification of the pfFasL (grey bars) and the sFasL (black bars) fractions, following transfection of the pfFasL plasmid in the presence of the indicated proportion of the sFasL plasmid. The measures were normalized to the condition lacking sFasL. Mean+/- sd from three experiments. Panel C: The TCR-pfFasL chimera (2 µg, according to an ELISA specific for the TCR-pFasL molecule using anti-TCRδ5 (clone 12C7) and anti-FasL (clone 10F2) as capture and tracing antibodies, respectively), produced in the absence or the presence of the sFasL plasmid at the indicated ratio, was immunoprecipitated with the anti-TCRδ5 antibody, then separated by 10% SDS-PAGE under reducing conditions and revealed by immunoblotting with the anti-FasL antibody. As a control, the immunoprecipitation experiment was performed with 2 µg of sFasL protein. Panel D: COS supernatants containing pfFasL (4 µg/ml according to the Flag ELISA) produced alone, was mixed with culture medium or sFasL (15 µg/ml) produced separately in a total volume of 1 ml, and incubated for 24 h at 37°C. Then the recombinant proteins were immunoprecipitated (left panels) with the anti-FasL (upper panel) or anti-Flag (lower panel) antibodies, followed by a SDS-PAGE under reducing conditions and immunoblotting with an anti-FasL antibody. As a control, the same experiment was performed for the sFasL molecule (15 µg according to the FasL ELISA, right panel).

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Produced, Plasmid Preparation, Immunoprecipitation, SDS Page, Transfection, Incubation, Recombinant

    Effect of sFasL on the supernatant production of the Flag-tagged FasL constructs. Panels A to D : An increasing amount expressed in percentage, of the sFasL encoding plasmid, was co-transfected with a fixed amount of the plasmids encoding sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D). The secreted proteins were quantified in culture supernatants using an ELISA specific for FasL (shaded histograms, right-hand scale) and for Flag-tagged FasL (curves, left-hand scale). For the Flag ELISA, the measured concentrations were normalized according to the condition lacking sFasL. Are presented the mean +/- sd of four independent transfection experiments. * 0.02≤p≤0.05; ** p≤0.02. Panel E : direct anti-FasL immunoblot analysis of identical volumes of the cell culture supernatant containing pfFasL produced alone and with 50% of the sFasL plasmid, after SDS-PAGE separation under reducing conditions.

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Effect of sFasL on the supernatant production of the Flag-tagged FasL constructs. Panels A to D : An increasing amount expressed in percentage, of the sFasL encoding plasmid, was co-transfected with a fixed amount of the plasmids encoding sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D). The secreted proteins were quantified in culture supernatants using an ELISA specific for FasL (shaded histograms, right-hand scale) and for Flag-tagged FasL (curves, left-hand scale). For the Flag ELISA, the measured concentrations were normalized according to the condition lacking sFasL. Are presented the mean +/- sd of four independent transfection experiments. * 0.02≤p≤0.05; ** p≤0.02. Panel E : direct anti-FasL immunoblot analysis of identical volumes of the cell culture supernatant containing pfFasL produced alone and with 50% of the sFasL plasmid, after SDS-PAGE separation under reducing conditions.

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Construct, Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Produced, SDS Page

    Effect of sFasL on cell targeting of the FasL-containing chimeras. Panel A : Schematic description of the experimental model used. The chimera is enriched at the surface of the CD32-expressing L-cells via its HLA targeting module and an anti-HLA monoclonal antibody. Panel B: murine Fas (continuous line), human CD32 (dashed line) and IgG1 isotype-matched control (shaded histogram) staining of the CD32+ L-cell transfectant. Living cells were gated on the basis of the morphological parameters. Panel C : Fas sensitivity of the CD32+ L-cell transfectant to the indicated concentrations of the anti-Fas JO-2 antibody (circles), the HLA-pfFasL chimera expressed alone (triangle) or in the presence of 25% of the sFasL plasmid (squares), in the MTT viability assay. Panel D : The CD32+ L-cells were incubated with the HLA-pfFasL chimera produced in the presence (black bars) or in the absence (white bars) of 25% of the sFasL plasmid, together with the indicated irrelevant IgG1 isotype-matched, anti-beta-2 microglobulin or anti-Flag antibodies. The concentrations of the chimera that triggered 15% of cell death and were at 15 and 0.3 ng/ml in the absence and presence of sFasL, as estimated using the ELISA specific for the Flag-tagged FasL. Cytotoxicity was measured with the propidium iodide assay and normalized to the effect of the chimera in the absence of antibody. Are presented the mean +/- sd of three independent experiments. Panel E: reversal in the presence of the blocking anti-FasL and anti-CD32 antibodies, of the cytotoxic effect of the immune complexes between the anti-Flag antibody and HLA-pfFasL co-expressed with sFasL. Are presented the mean +/- sd of three independent experiments. ns : non significant ; ** p≤0.02.

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Effect of sFasL on cell targeting of the FasL-containing chimeras. Panel A : Schematic description of the experimental model used. The chimera is enriched at the surface of the CD32-expressing L-cells via its HLA targeting module and an anti-HLA monoclonal antibody. Panel B: murine Fas (continuous line), human CD32 (dashed line) and IgG1 isotype-matched control (shaded histogram) staining of the CD32+ L-cell transfectant. Living cells were gated on the basis of the morphological parameters. Panel C : Fas sensitivity of the CD32+ L-cell transfectant to the indicated concentrations of the anti-Fas JO-2 antibody (circles), the HLA-pfFasL chimera expressed alone (triangle) or in the presence of 25% of the sFasL plasmid (squares), in the MTT viability assay. Panel D : The CD32+ L-cells were incubated with the HLA-pfFasL chimera produced in the presence (black bars) or in the absence (white bars) of 25% of the sFasL plasmid, together with the indicated irrelevant IgG1 isotype-matched, anti-beta-2 microglobulin or anti-Flag antibodies. The concentrations of the chimera that triggered 15% of cell death and were at 15 and 0.3 ng/ml in the absence and presence of sFasL, as estimated using the ELISA specific for the Flag-tagged FasL. Cytotoxicity was measured with the propidium iodide assay and normalized to the effect of the chimera in the absence of antibody. Are presented the mean +/- sd of three independent experiments. Panel E: reversal in the presence of the blocking anti-FasL and anti-CD32 antibodies, of the cytotoxic effect of the immune complexes between the anti-Flag antibody and HLA-pfFasL co-expressed with sFasL. Are presented the mean +/- sd of three independent experiments. ns : non significant ; ** p≤0.02.

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Expressing, Staining, Transfection, Plasmid Preparation, MTT Assay, Viability Assay, Incubation, Produced, Enzyme-linked Immunosorbent Assay, Blocking Assay

    Effect of sFasL on the cytotoxic activity of the Flag-tagged FasL chimeras. The FasL-derived proteins sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D) were expressed alone or upon co-transfection with the indicated percentage of the plasmid encoding sFasL. A fixed concentration triggering 25 to 40% of cell death (1.9 ng/ml for sfFasL, 0.6 ng/ml for pfFasL, 0.7 ng/ml for HLA-pfFasL and 2.2 ng/ml for TCR-pfFasL), for the FasL-derived protein quantitated with the ELISA specific for Flag-tagged FasL, was incubated with the Fas-sensitive Jurkat cells. For the sfFasL construct, the filled squares and the empty squares depict the cytotoxicity of sfFasL in the presence and absence of the cross-linking anti-Flag antibody at 0.5 µg/ml), respectively. Cytotoxicity was estimated by a measure of the remaining viable cells using the MTT assay. Are presented the mean +/- sd of four independent transfection experiments. * 0.01≤p≤0.05; ** p≤0.01.

    Journal: PLoS ONE

    Article Title: Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

    doi: 10.1371/journal.pone.0073375

    Figure Lengend Snippet: Effect of sFasL on the cytotoxic activity of the Flag-tagged FasL chimeras. The FasL-derived proteins sfFasL (Panel A), pfFasL (Panel B), TCR-pfFasL (Panel C) and HLA-pfFasL (Panel D) were expressed alone or upon co-transfection with the indicated percentage of the plasmid encoding sFasL. A fixed concentration triggering 25 to 40% of cell death (1.9 ng/ml for sfFasL, 0.6 ng/ml for pfFasL, 0.7 ng/ml for HLA-pfFasL and 2.2 ng/ml for TCR-pfFasL), for the FasL-derived protein quantitated with the ELISA specific for Flag-tagged FasL, was incubated with the Fas-sensitive Jurkat cells. For the sfFasL construct, the filled squares and the empty squares depict the cytotoxicity of sfFasL in the presence and absence of the cross-linking anti-Flag antibody at 0.5 µg/ml), respectively. Cytotoxicity was estimated by a measure of the remaining viable cells using the MTT assay. Are presented the mean +/- sd of four independent transfection experiments. * 0.01≤p≤0.05; ** p≤0.01.

    Article Snippet: The anti-FasL 14C2 or the anti-Flag mAbs were pre-coated overnight onto 96 well ELISA plates (Maxisorp Nunc, Thermo Scientific, Rochester, USA) respectively at 1 µg or 0.25 µg/well in hydrogenocarbonate coating buffer (pH = 9.6).

    Techniques: Activity Assay, Derivative Assay, Cotransfection, Plasmid Preparation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Construct, MTT Assay, Transfection

    Analysis of GRP78–HTJ1 and OxPL–GRP78 interactions. (A) GRP78 and HTJ1 expression in HPAEC and human lung microvascular endothelial cells was detected by Western Blot. (B, C) GRP78 interactions were analyzed in coimmunoprecipitation assays using lysates from control or DMPC- (10 μg/ml, 15 min) or OxPAPC-stimulated (10 μg/ml) cells with antibody to GRP78 (B, top), HTJ1 (B, bottom), or EO6 antibody recognizing OxPL (C). (D) Human recombinant GRP78 was incubated with OxPAPC, OxPAPS, or their oxidation-resistant analogues DMPC or DMPS. Left, native gel electrophoresis, followed by Western blot with anti-GRP78 antibody. Shift in electrophoretic mobility of GRP78 incubated with OxPAPS, but not DMPS, indicates formation of GRP78–OxPAPS complex. Right, SDS–PAGE, followed by Western blot with EO6 antibody and reprobing with anti-GRP78 antibody. Positive EO6 immunoreactivity of GRP78 preincubated with OxPAPC indicates formation of GRP78–OxPAPC complex. (E) ELISA plates coated with OxPAPS or DMPS or control uncoated plates incubated with PBS incubated with human recombinant GRP78 (left) or HPAEC lysates (middle and right). The bound GRP78 was detected using anti-GRP78 antibody. * p

    Journal: Molecular Biology of the Cell

    Article Title: GRP78 is a novel receptor initiating a vascular barrier protective response to oxidized phospholipids

    doi: 10.1091/mbc.E13-12-0743

    Figure Lengend Snippet: Analysis of GRP78–HTJ1 and OxPL–GRP78 interactions. (A) GRP78 and HTJ1 expression in HPAEC and human lung microvascular endothelial cells was detected by Western Blot. (B, C) GRP78 interactions were analyzed in coimmunoprecipitation assays using lysates from control or DMPC- (10 μg/ml, 15 min) or OxPAPC-stimulated (10 μg/ml) cells with antibody to GRP78 (B, top), HTJ1 (B, bottom), or EO6 antibody recognizing OxPL (C). (D) Human recombinant GRP78 was incubated with OxPAPC, OxPAPS, or their oxidation-resistant analogues DMPC or DMPS. Left, native gel electrophoresis, followed by Western blot with anti-GRP78 antibody. Shift in electrophoretic mobility of GRP78 incubated with OxPAPS, but not DMPS, indicates formation of GRP78–OxPAPS complex. Right, SDS–PAGE, followed by Western blot with EO6 antibody and reprobing with anti-GRP78 antibody. Positive EO6 immunoreactivity of GRP78 preincubated with OxPAPC indicates formation of GRP78–OxPAPC complex. (E) ELISA plates coated with OxPAPS or DMPS or control uncoated plates incubated with PBS incubated with human recombinant GRP78 (left) or HPAEC lysates (middle and right). The bound GRP78 was detected using anti-GRP78 antibody. * p

    Article Snippet: GRP78 ELISA Microtiter 96-well plates (MaxiSorp; Nunc, Thermo Scientific, Rochester, NY) were coated with OxPAPS or DMPS (each 100 μg/ml in PBS containing 0.01% BHT) at 4°C overnight.

    Techniques: Expressing, Western Blot, Recombinant, Incubation, Nucleic Acid Electrophoresis, SDS Page, Enzyme-linked Immunosorbent Assay