Journal: PLoS ONE

Article Title: Apolipoprotein J/Clusterin in Human Erythrocytes Is Involved in the Molecular Process of Defected Material Disposal during Vesiculation

doi: 10.1371/journal.pone.0026033

Figure Lengend Snippet: (A, B) TEM immunogold localization of sCLU in RBCs membrane protrusions (A) and vesicles (ves) (B) collected from fresh units of stored RBCs (N = 2, young healthy donors). Solid or dashed arrows indicate sCLU immunogold localization at the periphery or the cytosol of the vesicles, respectively. (C) Representative immunoblot analysis of RBCs-derived purified vesicles (N = 2) probed with either polyclonal anti-sCLU or with monoclonal anti-Band 3 antibodies. Molecular weight markers are indicated to the right of the blot. Bars in (A), (B), 100 nm.

Article Snippet: The polyclonal antibody against CD59 was obtained from R&D Systems (AF 1987).

Techniques: Membrane, Western Blot, Derivative Assay, Purification, Molecular Weight

Journal: PLoS ONE

Article Title: Apolipoprotein J/Clusterin in Human Erythrocytes Is Involved in the Molecular Process of Defected Material Disposal during Vesiculation

doi: 10.1371/journal.pone.0026033

Figure Lengend Snippet: Purified RBCs membranes from healthy subjects (N = 6) were lysed in NP-40 and lysates were immunoprecipitated (IP) with polyclonal antibodies against sCLU, Band 3, stomatin or normal serum (control). Immunoprecipitates were immunoblotted (IB) under reducing conditions for sCLU (A 1 , upper panel), Band 3 (A 1 , middle panel), CD59 (A 1 , lower panel) and Hb (A 3 ); shown IPs are representatives from two independent experiments. (A 2 ) CLSM co-immunolocalization of the sCLU and Band 3 proteins at the RBCs plasma membrane. Cells were co-stained with anti-Band 3 monoclonal (green; upper panel) and anti-sCLU polyclonal antibodies (red; lower panel). Captured images were merged to reveal co-distribution sites (yellow; lower panel, arrows). Bars, 3 µm. (B) Anti-dinitrophenylhydrazone (DNP) immunoblotting of sCLU, Band 3, and control (IgGs) immunoprecipitates for the detection of co-immunoprecipitated carbonylated proteins (arrows) in 2,4-dinitrophenylhydrazine-modified (OX) or unmodified protein material.

Article Snippet: The polyclonal antibody against CD59 was obtained from R&D Systems (AF 1987).

Techniques: Purification, Immunoprecipitation, Control, Clinical Proteomics, Membrane, Staining, Western Blot, Modification

Journal: PLoS ONE

Article Title: Apolipoprotein J/Clusterin in Human Erythrocytes Is Involved in the Molecular Process of Defected Material Disposal during Vesiculation

doi: 10.1371/journal.pone.0026033

Figure Lengend Snippet: Erythrocytic sCLU localizes at both sides of the plasma membrane in association with non-cytoskeletal areas, as well as in the cytosol (see also, Antonelou et al., accompanying paper). At the intracellular side of the RBCs membrane sCLU may bind Band 3, Hb and/or other cytoskeleton-free membrane portions. On the other hand, the sCLU that localizes at the extracellular side of the RBCs membrane can attach to membrane by binding to Band 3, CD59, plasma membrane IgGs or to an currently unknown sCLU-specific receptor. Physiological in vivo or ex vivo RBCs senescence (1) is associated with cytosol, cytoskeleton and membrane structural alterations, including Band 3 modifications, increased membrane binding of IgGs, proteolysis, protein aggregation and increased oxidation defects. Vesiculation (2), a self-protective mechanism of mammalian erythrocytes, removes oxidized proteins and aggregates from both plasma membrane and cytosol thereby postponing the untimely elimination of otherwise healthy erythrocytes. This process takes place through the entire in vivo or ex vivo lifespan of RBCs and is functionally connected to the release of sCLU-, Band 3-, CD59-, Hb- and IgGs-containing vesicles. We propose that vesicular sCLU by following its membrane linkers (e.g. Band 3) or other unknown cytosolic interacting proteins assists via its chaperone function in the disposal of non-functional or death signalling effective material from RBCs.

Article Snippet: The polyclonal antibody against CD59 was obtained from R&D Systems (AF 1987).

Techniques: Clinical Proteomics, Membrane, Binding Assay, In Vivo, Ex Vivo, Functional Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Regulation of Complement-Dependent Cytotoxicity by MicroRNAs miR-200b, miR-200c, and miR-217.

doi: 10.4049/jimmunol.1502701

Figure Lengend Snippet: FIGURE 2. miR-200 (b and c) regulates cell sensitivity to complement, C5b-9 deposition, and CD46 and CD55 expression. (A) K562 cells were transfected with miR-200b/c or control plasmid. After 3 or 24 h, cells were treated with Ab and NHS. Cell death (%) was measured by trypan blue inclusion. (B) K562 cells were transfected with miRNA inhibitors specific for miR-200b and miR-200c, or with nonspecific oligonucleotide as negative control. After 24 h, cells were treated with Ab and complement, as above. Cell death (%) was measured by propidium iodide inclusion. (C) K562 cells transfected with miR-200b/c or a control plasmid for 24 h were treated with a sublytic dose of Ab and then with NHS for 10 min at 37˚C. Then they were labeled with goat anti-C3 Ab and FITC-conjugated secondary Ab and analyzed by flow cytometry. Mean fluorescence intensity (MFI) values of cell-bound C3b, representative of three independent experiments, are shown. (D) K562 cells transfected with miR-200b/c or a control plasmid for 24 h were treated with sublytic complement, as in (C). Then they were labeled with mouse anti– C5b-9 aE-11 Ab and FITC-conjugated secondary Ab and analyzed by flow cytometry. MFI values of cell-bound C5b-9, representative of three independent experiments, are shown. (E) K562 cells transfected for 24 h with inhibitors of miR-200b and miR-200c, or with nonspecific oligonucleotide, were incubated with sublytic Ab and NHS for 10 min at 37˚C. The cells were labeled with the aE-11 Ab, as above. MFI values of cell-bound C5b-9, representative of three independent experiments, are shown. (F) K562 cells were transfected with miR-200b/c or control plasmid for 24 h and were labeled with mouse anti-CD46, anti-CD55, or anti- CD59 Ab and then with FITC-conjugated secondary Ab. Cells were then analyzed by flow cytometry, and MFI values were determined. Results, representative of three independent experiments, present each regulator’s level relative to its expression in control cells (set as 100%). *p , 0.05, **p , 0.01 relative to control.

Article Snippet: Mouse anti-human CD46, anti-human CD55, and anti-human CD59 mAbs were purchased from AbD Serotec (Oxford, U.K.).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Negative Control, Labeling, Cytometry, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Regulation of Complement-Dependent Cytotoxicity by MicroRNAs miR-200b, miR-200c, and miR-217.

doi: 10.4049/jimmunol.1502701

Figure Lengend Snippet: FIGURE 3. miR-217 regulates cell sensitivity to complement, C3 and C5b-9 deposition, and CD46 expression. (A) K562 cells were transfected with miR-217 or control plasmid. After 3 or 24 h, cells were treated with Ab and NHS (60 min). Cell death (%) was measured by trypan blue inclusion. (B) K562 cells were transfected with miRNA inhibitors specific for miR-217 or with nonspecific oligonucleotide as negative control. After 24 h, cells were treated with Ab and complement, as above. Cell death (%) was measured by propidium iodide inclusion. (C and D) K562 cells transfected for 24 h with miR-217 or control plasmid were incubated with a sublytic dose of Ab and NHS for 10 min at 37˚C. Cells were labeled with goat anti-C3b Ab and FITC-conjugated secondary Ab (C) or mouse anti–C5b-9 aE-11 Ab and FITC-conjugated secondary Ab (D) and analyzed by flow cytometry. Mean fluorescence intensity (MFI) values of cell-bound C3b or C5b-9, representative of three independent experiments, are shown. (E) K562 cells transfected for 24 h with miR-217 inhibitor or with nonspecific oligonucleotide (C) were incubated with a sublytic dose of Ab and NHS for 10 min at 37˚C. The cells were labeled with aE-11 Ab and FITC-conjugated secondary Ab, and MFI values of cell-bound C5b-9 were determined. Results are representative of three independent experiments. (F) K562 cells were transfected with miR-217 or control plasmid for 24 h and were then labeled with mouse anti-CD46, anti-CD55, or anti-CD59 Ab and FITC-conjugated secondary Ab. Cells were then analyzed by flow cytometry, and MFI values were determined. Results, representative of three independent experiments, present each regulator’s level relative to its expression in control cells (set as 100%). *p , 0.05, **p , 0.01 relative to control.

Article Snippet: Mouse anti-human CD46, anti-human CD55, and anti-human CD59 mAbs were purchased from AbD Serotec (Oxford, U.K.).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Negative Control, Incubation, Labeling, Cytometry

Journal: Cancer research

Article Title: p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

doi: 10.1158/0008-5472.CAN-05-3191

Figure Lengend Snippet: Figure 1. p53 binds to the putative binding sites within the CD59 gene in vitro. A, CD59.1, the first putative p53-responsive element, is located in the 5V flanking region of the CD59 gene ranging from nucleotide 1665 to 1637 upstream of exon 1. CD59.2 is the second putative p53-binding sequence located in intron 1 of CD59 ranging from nucleotide 675 to 645 upstream of exon 2. Possible p53 half-binding sites are marked in capital letters. The differences between these and the p53 consensus binding sequence are underlined. B, EMSA with samples labelled with horseradish peroxidase, incubated with 100 ng wild-type recombinant p53 in the presence of 0.1 Ag/AL poly(deoxyinosinic-deoxycytidylic acid). Oligonucleotides were separated in a 2% agarose gel and detected by autoradiography.

Article Snippet: Primary antibodies used were mouse monoclonal BRIC229 (International Blood Group Reference Laboratory, Bristol, United Kingdom) for detection of CD59; rabbit polyclonal anti-acetyl-p53(Lys373, Lys382) and mouse monoclonal anti-p53, clone BP53-12 (Upstate, Dundee, United Kingdom) for detection of acetylated and total p53, respectively; rabbit polyclonal antip42 mitogen-activated protein kinase (MAPK; Cell Signaling Technology) for p42 MAPK detection.

Techniques: Binding Assay, In Vitro, Sequencing, Incubation, Recombinant, Agarose Gel Electrophoresis, Autoradiography

Journal: Cancer research

Article Title: p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

doi: 10.1158/0008-5472.CAN-05-3191

Figure Lengend Snippet: Figure 2. p53-dependent expression of CD59 in HeLa cells. A, Western blot analysis of p53 (anti-p53, cloneBP53-12 recognizes all p53 modifications) and CD59 expression in HeLa cells and cells 36 hours after transfection with siRNA specifically blocking expression of p53. Detection of p42 MAPK was done as a control for the siRNA specificity. B, fluorescence-activated cell sorting analysis of CD59 expression in HeLa and p53 siRNA–transfected cells.

Article Snippet: Primary antibodies used were mouse monoclonal BRIC229 (International Blood Group Reference Laboratory, Bristol, United Kingdom) for detection of CD59; rabbit polyclonal anti-acetyl-p53(Lys373, Lys382) and mouse monoclonal anti-p53, clone BP53-12 (Upstate, Dundee, United Kingdom) for detection of acetylated and total p53, respectively; rabbit polyclonal antip42 mitogen-activated protein kinase (MAPK; Cell Signaling Technology) for p42 MAPK detection.

Techniques: Expressing, Western Blot, Transfection, Blocking Assay, Control, Fluorescence, FACS

Journal: Cancer research

Article Title: p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

doi: 10.1158/0008-5472.CAN-05-3191

Figure Lengend Snippet: Figure 3. Alterations in CD59 expression in IMR32 cells treated with camptothecin. A, Western blot analysis of CD59 expression in IMR32 cells treated with camptothecin for either 24 or 48 hours. B, expression of CD59 in IMR32 cells treated with camptothecin for 24 or 48 hours detected by quantitative PCR. Expression in untreated cells is set as 100%. Columns, mean for two independent experiments; bars, SD. Compared sets are shown by columns with interrelated Ps for comparison. C, resistance of IMR32 (x) and surviving IMR32 (n) cells to C lysis. Lysis assay with preincubation of both untreated (.) and surviving (E) IMR32 cells with CD59-blocking antibody BRIC229 was carried out as a control. Points, mean for three independent experiments; bars, SD.

Article Snippet: Primary antibodies used were mouse monoclonal BRIC229 (International Blood Group Reference Laboratory, Bristol, United Kingdom) for detection of CD59; rabbit polyclonal anti-acetyl-p53(Lys373, Lys382) and mouse monoclonal anti-p53, clone BP53-12 (Upstate, Dundee, United Kingdom) for detection of acetylated and total p53, respectively; rabbit polyclonal antip42 mitogen-activated protein kinase (MAPK; Cell Signaling Technology) for p42 MAPK detection.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Comparison, Lysis, Blocking Assay, Control

Journal: Cancer research

Article Title: p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

doi: 10.1158/0008-5472.CAN-05-3191

Figure Lengend Snippet: Figure 4. Expression of p53 and recruitment to the CD59 gene. A, two different antibodies were used for detection of p53: anti-p53, clone BP53-12 recognizing all forms of p53 and anti–acetyl-p53 (Lys373, Lys382) to detect the changes in p53 acetylation. B, IMR32 cells were treated with camptothecin for either 24 or 48 hours. Immunoprecipitation was done with anti–acetyl-p53 (Lys373, Lys382) antibody. Binding of p53 to its responsive elements CD59.1 (black columns) and CD59.2 (striped columns) in control IMR32 cells is set as 1. Columns, mean for two independent chromatin immunoprecipitation experiments each analyzed in duplicate; bars, SD. Compared sets are shown by columns with interrelated Ps for comparison.

Article Snippet: Primary antibodies used were mouse monoclonal BRIC229 (International Blood Group Reference Laboratory, Bristol, United Kingdom) for detection of CD59; rabbit polyclonal anti-acetyl-p53(Lys373, Lys382) and mouse monoclonal anti-p53, clone BP53-12 (Upstate, Dundee, United Kingdom) for detection of acetylated and total p53, respectively; rabbit polyclonal antip42 mitogen-activated protein kinase (MAPK; Cell Signaling Technology) for p42 MAPK detection.

Techniques: Expressing, Immunoprecipitation, Binding Assay, Control, Chromatin Immunoprecipitation, Comparison

Journal: Cancer research

Article Title: p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

doi: 10.1158/0008-5472.CAN-05-3191

Figure Lengend Snippet: Figure 6. Chromatin immunoprecipitation analysis of recruitment of p53 to the CD59 gene in living Hep3B cells. Cells were treated with IFN-g or IL-8 for 36 hours. Immunoprecipitation was carried out either with anti–acetyl-p53 (Lys373, Lys382; A) or anti-p53, clone BP53-12 (B) antibodies. Binding of p53 to its responsive elements CD59.1 (black columns) and CD59.2 (white columns) in untreated cells is set as 1. Columns, mean for two independent chromatin immunoprecipitation experiments each analyzed in duplicate; bars, SD. Compared sets are shown by columns with interrelated Ps for comparison.

Article Snippet: Primary antibodies used were mouse monoclonal BRIC229 (International Blood Group Reference Laboratory, Bristol, United Kingdom) for detection of CD59; rabbit polyclonal anti-acetyl-p53(Lys373, Lys382) and mouse monoclonal anti-p53, clone BP53-12 (Upstate, Dundee, United Kingdom) for detection of acetylated and total p53, respectively; rabbit polyclonal antip42 mitogen-activated protein kinase (MAPK; Cell Signaling Technology) for p42 MAPK detection.

Techniques: Chromatin Immunoprecipitation, Immunoprecipitation, Binding Assay, Comparison

Journal: Cancer research

Article Title: p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

doi: 10.1158/0008-5472.CAN-05-3191

Figure Lengend Snippet: Figure 5. Effect of cytokines on p53 and CD59 expression in IMR32 and Hep3B cells. A, quantitative PCR analysis of p53 (black columns) and CD59 (white columns) expression following an incubation of Hep3B and HL60 cells with IFN-g or IL-8. Results from two independent measurements displayed a significant difference between untreated and cytokine treated Hep3B samples. *, P < 0.05; **, P < 0.01. However, no significant difference was observed between HL60 samples. B, flow cytometry analysis of p53 (black columns; Lys373, Lys382) acetylated p53 (white columns) and CD59 (striped columns) following the same treatments as in (A). *, P < 0.01; **, P < 0.001. Columns, mean of four measurements obtained from two separate experiments; bars, SD.

Article Snippet: Primary antibodies used were mouse monoclonal BRIC229 (International Blood Group Reference Laboratory, Bristol, United Kingdom) for detection of CD59; rabbit polyclonal anti-acetyl-p53(Lys373, Lys382) and mouse monoclonal anti-p53, clone BP53-12 (Upstate, Dundee, United Kingdom) for detection of acetylated and total p53, respectively; rabbit polyclonal antip42 mitogen-activated protein kinase (MAPK; Cell Signaling Technology) for p42 MAPK detection.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Incubation, Flow Cytometry

Journal:

Article Title: Human lung cancer cell lines express cell membrane complement inhibitory proteins and are extremely resistant to complement-mediated lysis; a comparison with normal human respiratory epithelium in vitro , and an insight into mechanism(s) of resistance

doi: 10.1046/j.1365-2249.1998.00581.x

Figure Lengend Snippet: Cell membrane expression of complement inhibitory protein (CIP) molecules [membrane cofactor protein (MCP), decay-accelerating factor (DAF), CD59] as assessed by flow cytometry of human lung cancer cell lines (non-small cell bronchogenic carcinoma) and of normal nasal epithelial cells in primary cultures.

Article Snippet: Rat anti-human CD59 MoAb YTH 53.1 (IgG2b) was purchased from Serotec (Oxford, UK) [ 12 ].

Techniques: Expressing, Flow Cytometry

Journal:

Article Title: Human lung cancer cell lines express cell membrane complement inhibitory proteins and are extremely resistant to complement-mediated lysis; a comparison with normal human respiratory epithelium in vitro , and an insight into mechanism(s) of resistance

doi: 10.1046/j.1365-2249.1998.00581.x

Figure Lengend Snippet: Effect of neutralizing antibodies against cell membrane complement inhibitory protein (CIP) molecules [anti-membrane cofactor protein (MCP), GB-24, decay-accelerating factor (DAF), IA-10, anti-CD59, YTH 53.1 or antiserum as indicated] on complement-mediated cell lysis of lung cancer cell lines (a) and of normal nasal epithelial cells (NEC) (b). Cells were incubated with antibodies (1:50 dilution, 30 min, at 4°C), then washed and exposed to 50% normal human serum (NHS). In some experiments cells were presensitized with anti-carcinoembryonic antigen (CEA) or with anti-NEC (1:20 dilution, each). Values are mean ± s.d. of four to six experiments. (a) *P < 0.01 versus lysis of same cells with NHS and no antibodies or versus lysis with anti-CD59; **P < 0.025 versus lysis of same cells with NHS and no antibodies. (b) **P < 0.001 versus lysis of same cells with NHS and no antibodies (Student's t-test for all comparisons). Data for normal NEC, except for the effect of anti-CD59, YTH 53.1, were obtained from our recent concomitant study [5].

Article Snippet: Rat anti-human CD59 MoAb YTH 53.1 (IgG2b) was purchased from Serotec (Oxford, UK) [ 12 ].

Techniques: Lysis, Incubation

Journal:

Article Title: Human lung cancer cell lines express cell membrane complement inhibitory proteins and are extremely resistant to complement-mediated lysis; a comparison with normal human respiratory epithelium in vitro , and an insight into mechanism(s) of resistance

doi: 10.1046/j.1365-2249.1998.00581.x

Figure Lengend Snippet: Detachment of decay-accelerating factor (DAF) and CD59 from the NCI-H596 lung cancer cell line cell membrane by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PIPLC; 0.5 U/ml, 37°C, 45 min). Expression of cell membrane DAF and CD59 was assayed by flow cytometry. Results for ChaGo K-1 cells were the same (not shown).

Article Snippet: Rat anti-human CD59 MoAb YTH 53.1 (IgG2b) was purchased from Serotec (Oxford, UK) [ 12 ].

Techniques: Expressing, Flow Cytometry

Journal:

Article Title: Human lung cancer cell lines express cell membrane complement inhibitory proteins and are extremely resistant to complement-mediated lysis; a comparison with normal human respiratory epithelium in vitro , and an insight into mechanism(s) of resistance

doi: 10.1046/j.1365-2249.1998.00581.x

Figure Lengend Snippet: Effect of decay-accelerating factor (DAF) and CD59 detachment from the cell membrane of lung cancer cell lines on the cells’ susceptibility to complement-mediated lysis. (a) Cells were treated first with phosphatidylinositol-specific phospholipase C (PIPLC; 45 min, 37°C) at various concentrations, then washed and exposed to 50% normal human serum (NHS; 60 min, 37°C). *P < 0.001 from the alternate preceding value; **P < 0.05 from preceding value for ChaGo K-1 cells; ***P < 0.01 from the alternate preceding value, for NCI-H596 cells. (b) Cells were first treated with PIPLC, then washed and exposed to various concentrations of NHS. **P < 0.001 from the preceding value for both cell types. Values are mean ± s.d. of four to six experiments (one-way anova for all comparisons).

Article Snippet: Rat anti-human CD59 MoAb YTH 53.1 (IgG2b) was purchased from Serotec (Oxford, UK) [ 12 ].

Techniques: Lysis

Journal: The Journal of Biological Chemistry

Article Title: Basigin Interacts with Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 as a Second Erythrocyte Receptor to Promote Parasite Growth *

doi: 10.1074/jbc.M116.744367

Figure Lengend Snippet: Identification of putative erythrocyte surface proteins interacting with PvTRAg38 by MudPIT analysis Proteins were identified after LC-MS/MS analysis following in solution digestion of GST-PvTRAg38 pulled down proteins.

Article Snippet: Materials The following were commercially obtained: human basigin cDNA clone, mammalian expressed recombinant histidine-tagged CD59 (Sino Biological, Beijing, China); human erythrocyte 55-kDa membrane protein (GeneCopoeia, Rockville, MD); Alexa Fluor 488-conjugated goat anti-mouse and anti-rabbit IgG, proofreading Pfx polymerase enzyme (Invitrogen Life Technologies, Inc.); sulfo-SBED-biotin Label transfer cross-linker (sulfosuccinimidyl-2-[6-(biotin amide)-2-( p -azidobenzamido)hexanoamido] ethyl-1,3′-dithiopropionate) (Thermo Scientific, Rockford, IL); GeneJET gel extraction kit (Thermo Scientific, Waltham, MA); HEK-293 cells (American Type Culture Collection (ATCC), Manassas, VA); RPMI 1640 medium, hypoxanthine, penicillin/streptomycin, fetal calf serum, glutamine, glutaraldehyde, poly- l -lysine, monoclonal antibodies against basigin (Clone MEM-M6); His 6 and GST (Sigma); Lipofectamine® 2000, synthetic peptides (Thermo Fisher Scientific, GmbH, Germany); mouse monoclonal antibodies DL6 (Santa Cruz Biotechnology, Dallas, TX); HBS-EP buffer (degassed and ready to use 0.01 m HEPES, pH 7.4, 0.15 m NaCl, 3 m m EDTA, 0.005% v/v surfactant P20); protein marker standards for GPC (GE Healthcare); Matchmaker Gold Systems (Clontech); and trypsin (Promega Corp., Madison, WI).

Techniques: Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Basigin Interacts with Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 as a Second Erythrocyte Receptor to Promote Parasite Growth *

doi: 10.1074/jbc.M116.744367

Figure Lengend Snippet: Binding of PvTRAg38 to basigin. A, solid phase ELISA. Increasing concentrations (0–2.5 μm) of histidine-tagged basigin, 55-kDa EMP, or CD59 were added to the wells of an ELISA plate already coated with 50 nm GST-tagged PvTRAg38 or untagged thioredoxin (inset). The plate was developed with anti-His6 monoclonal antibody as described in text. Mean ± S.D. value of absorbance from three experiments is plotted. B, SPR analysis of basigin interaction with PvTRAg38. The recombinant basigin was immobilized on the cell of CM5 chip by amine coupling method. Five different concentrations of recombinant PvTRAg38 (100–500 nm) were injected at a flow rate of 30 μl/min over the surface of immobilized basigin. Curve fit (Langmuir 1:1 model) sensograms show dose-dependent response of PvTRAg38 binding with basigin. C, direct interaction between basigin and PvTRAg38 by Label transfer assay. Recombinant histidine-tagged basigin was incubated with recombinant GST-tagged PvTRAg38 (labeled with trifunctional Sulfo-SBED cross-linker) and processed as in Fig. 1A. The eluate was washed and resolved on 15% SDS-PAGE and transferred to nitrocellulose membrane. The blot was probed with streptavidin-HRP. Lane 1, GST-tagged PvTRAg38 and histidine-tagged basigin after Label transfer. Lane 2, labeled GST-tagged PvTRAg38. Sizes of molecular weight marker proteins are shown on right-hand side. D, specificity of binding of PvTRAg38 to basigin by competition assay. Increasing concentrations of histidine-tagged PvTRAg38 (0–2 μm) were incubated for 2 h with recombinant-untagged basigin immobilized on an ELISA plate well. After washing, a fixed concentration of GST-tagged PvTRAg38 was added to the wells, and the bound protein was detected with GST monoclonal antibody as described in the text. Binding in the absence of histidine-PvTRAg38 was taken as percentage control for the rest of the concentrations. The mean value of three independent experiments is plotted with S.D. E, specificity of binding between basigin and PvTRAg38 by antibody inhibition assay using anti-PvTRAg38 antibodies. Different dilutions of anti-PvTRAg38 antibody were incubated with recombinant GST-tagged PvTRAg38 immobilized on an ELISA plate well for 2 h. After washing, a fixed concentration of histidine-tagged basigin was added to the wells. The bound histidine-tagged basigin was detected using monoclonal anti-His6 antibodies as described in the text. The mean value of three independent experiments is plotted with S.D. F, specificity of binding between basigin and PvTRAg38 by antibody inhibition assay using anti-basigin antibodies. Different dilutions of polyclonal anti-basigin antibody were incubated with recombinant histidine-tagged basigin immobilized on an ELISA plate well for 2 h. After washing, a fixed concentration of GST-tagged PvTRAg38 was added to the wells, and bound recombinant GST-tagged PvTRAg38 was detected by using anti-GST antibodies as described in the text. The mean value of three independent experiments is plotted with S.D.

Article Snippet: Materials The following were commercially obtained: human basigin cDNA clone, mammalian expressed recombinant histidine-tagged CD59 (Sino Biological, Beijing, China); human erythrocyte 55-kDa membrane protein (GeneCopoeia, Rockville, MD); Alexa Fluor 488-conjugated goat anti-mouse and anti-rabbit IgG, proofreading Pfx polymerase enzyme (Invitrogen Life Technologies, Inc.); sulfo-SBED-biotin Label transfer cross-linker (sulfosuccinimidyl-2-[6-(biotin amide)-2-( p -azidobenzamido)hexanoamido] ethyl-1,3′-dithiopropionate) (Thermo Scientific, Rockford, IL); GeneJET gel extraction kit (Thermo Scientific, Waltham, MA); HEK-293 cells (American Type Culture Collection (ATCC), Manassas, VA); RPMI 1640 medium, hypoxanthine, penicillin/streptomycin, fetal calf serum, glutamine, glutaraldehyde, poly- l -lysine, monoclonal antibodies against basigin (Clone MEM-M6); His 6 and GST (Sigma); Lipofectamine® 2000, synthetic peptides (Thermo Fisher Scientific, GmbH, Germany); mouse monoclonal antibodies DL6 (Santa Cruz Biotechnology, Dallas, TX); HBS-EP buffer (degassed and ready to use 0.01 m HEPES, pH 7.4, 0.15 m NaCl, 3 m m EDTA, 0.005% v/v surfactant P20); protein marker standards for GPC (GE Healthcare); Matchmaker Gold Systems (Clontech); and trypsin (Promega Corp., Madison, WI).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Injection, Incubation, Labeling, SDS Page, Molecular Weight, Marker, Competitive Binding Assay, Concentration Assay, Inhibition

Journal: iScience

Article Title: Chimpanzee adenovirus-mediated multiple gene therapy for age-related macular degeneration

doi: 10.1016/j.isci.2023.107939

Figure Lengend Snippet: In vivo expression of eGFP, PEDF, sFlt-1, and sCD59 following IVT administration of AdCs (A) Representative fundus images of the retina from individual mice following IVT injection of 1.5×10 9 vp and 7.5×10 9 vp AdC68-eGFP. The eGFP signal could be detected from 48 h to 35 days post-injection. The dotted circles represent the edge of mouse retina. (B–D) Assessment of PEDF, sFlt-1, and sCD59 mRNA expression in retina-choroid-sclera complexes isolated from 10 mice. In each mouse, one eye was injected with AdC68-PFC (five mice for 1.5×10 9 vp and five mice for 7.5×10 9 vp) whereas the contralateral, un-injected eye served as control (only five eyes were used for analysis). At 4 days post-injection, RNA was purified from the retina-choroid complexes and real-time qPCR was conducted. Absolute number of mRNA copies were calculated using the standard curve method. (E–H) Images of western blot and quantification of the PEDF, sFlt-1, and sCD59 protein amount expressed in retina-choroid complexes of five mice. In each mouse, one eye was injected with AdC68-PFC (7.5×10 9 vp) whereas the contralateral, un-injected eye served as control. Total protein was obtained from retina-choroid-sclera complexes isolated from AdC68-PFC-treated (7.5×10 9 vp) and un-injected eyes 7 days post-injection. Antibodies against GAPDH were used for the internal control. The relative expression of PEDF, sFlt-1, and sCD59 in the un-injected eyes was set to 1. Data are expressed as mean ± SEM, and analyzed using one-way ANOVA multiple comparisons with Tukey’s method among groups in (B) and Student’s t test (two-tailed) in (C) (∗p < 0.05, ∗∗p < 0.01). PEDF, pigment epithelium-derived factor; sFlt-1, soluble fms-like tyrosine kinase-1; sCD59, soluble forms of CD59; IVT, intravitreal.

Article Snippet: PVDF membranes were blocked with 5% milk in PBST (PBS+Tween-20) for 2 h at RT and then target proteins were detected by specific primary antibodies including PEDF (1:500, 11104-RP02, Sino Biological, China), VEGFR1 (1:500, AF7748, Affinity, US), sCD59 (1:500, 12474-RP02, Sino Biological, China), Erk1/2 p44/42 (1:1000, 9102, Cell Signaling Technology, US), Erk1/2 phospho-p44/42 (T202/Y204) (1:1000, 9106, Cell Signaling Technology, US), p38 MAPK(1:1000, 8690, Cell Signaling Technology, US), Phospho-p38 MAPK (Thr180/Tyr182) (1:1000, 4631, Cell Signaling Technology, US), ICAM-1 (1:1000, 10831-1-AP, Proteintech, US), VCAM-1 (1:200, sc-13160 , Santa Cruz biotechnology, US).

Techniques: In Vivo, Expressing, Injection, Isolation, Control, Purification, Western Blot, Two Tailed Test, Derivative Assay

Journal: iScience

Article Title: Chimpanzee adenovirus-mediated multiple gene therapy for age-related macular degeneration

doi: 10.1016/j.isci.2023.107939

Figure Lengend Snippet:

Article Snippet: PVDF membranes were blocked with 5% milk in PBST (PBS+Tween-20) for 2 h at RT and then target proteins were detected by specific primary antibodies including PEDF (1:500, 11104-RP02, Sino Biological, China), VEGFR1 (1:500, AF7748, Affinity, US), sCD59 (1:500, 12474-RP02, Sino Biological, China), Erk1/2 p44/42 (1:1000, 9102, Cell Signaling Technology, US), Erk1/2 phospho-p44/42 (T202/Y204) (1:1000, 9106, Cell Signaling Technology, US), p38 MAPK(1:1000, 8690, Cell Signaling Technology, US), Phospho-p38 MAPK (Thr180/Tyr182) (1:1000, 4631, Cell Signaling Technology, US), ICAM-1 (1:1000, 10831-1-AP, Proteintech, US), VCAM-1 (1:200, sc-13160 , Santa Cruz biotechnology, US).

Techniques: Virus, Recombinant, Transfection, Protease Inhibitor, Plasmid Preparation, Gel Extraction, Software