Journal: Journal of Virology

Article Title: Identification of CD46 Binding Sites within the Adenovirus Serotype 35 Fiber Knob

doi: 10.1128/jvi.01732-07

Figure Lengend Snippet: FIG. 1. Analysis of recombinant Ad35 knob. (A) Western blot analysis. Purified Ad35 knob was run as native (N) or denatured protein (D) on a polyacrylamide gel. Coomassie brilliant blue staining (CBB) revealed the trimeric knob form (T), which is converted into monomers after boiling (M). Blots were analyzed for CD46 binding by subsequent incubation with sCD46, anti-CD46 MAb, and anti-mouse IgG-HRP (middle panel). After stripping, the same filters were incu- bated with an anti-His6-HRP antibody (right panel). (B) Kinetic re- sponse data for sCD46 binding to biosensor surface containing Ad35 knob (50 RU). Experimental data (black) represent the responses of duplicate injections of various concentrations of sCD46 (indicated above the corresponding curves). Global fits of these data according to a 1:1 interaction model are shown in red (see text for resulting kinetic constants).

Article Snippet: The blot was then incubated with anti-CD46 antibody (clone J4.48; Fitzgerald, Concord, MA) (1:50) in TBS and 3% milk for 1 h at RT and then washed three times for 10 min in TBS-T buffer.

Techniques: Recombinant, Western Blot, Staining, Binding Assay, Incubation, Stripping Membranes

Journal: Journal of Virology

Article Title: Identification of CD46 Binding Sites within the Adenovirus Serotype 35 Fiber Knob

doi: 10.1128/jvi.01732-07

Figure Lengend Snippet: FIG. 2. Library identification of amino acids involved in Ad35- CD46 binding. (A) Amino acid alignment of the Ad11 and Ad35 fiber knob sequences. Numbers represent the fiber amino acid sequences for Ad11p (GenBank accession AAN62521) and Ad35 (GenBank ac- cession AP_000601). -Sheet regions (A to J) are shown. Mutated amino acids from the Ad35 fiber knob that decrease CD46 binding are shown by green lines, and contact amino acids between the Ad11 fiber and CD46 (18) are shown by black lines.

Article Snippet: The blot was then incubated with anti-CD46 antibody (clone J4.48; Fitzgerald, Concord, MA) (1:50) in TBS and 3% milk for 1 h at RT and then washed three times for 10 min in TBS-T buffer.

Techniques: Binding Assay

Journal: Journal of Virology

Article Title: Identification of CD46 Binding Sites within the Adenovirus Serotype 35 Fiber Knob

doi: 10.1128/jvi.01732-07

Figure Lengend Snippet: FIG. 3. Analysis of functional properties of selected Ad35 knob mutants. (A) Binding of Arg279Cys mutant to sCD46 in Western blot analysis. Purified Ad35 wild-type and mutant knobs were run as native (N) or as denatured (D) protein on a polyacrylamide gel. Coomassie brilliant blue staining (CBB) revealed the trimeric knob form (T), which is converted into monomers after boiling (M). Blots were ana- lyzed for CD46 binding by subsequent incubation with sCD46, anti- CD46 MAb, and anti-mouse IgG-HRP. (B) Biacore response data for sCD46 binding to biosensor surface containing Arg297Cys knob (127 RU). Experimental data represent the responses of duplicate injec- tions of various concentrations of sCD46 (169 nM, 56 nM, 19 nM, 6 nM, and 2 nM). Experimental conditions were the same as for wild- type Ad35 knob (Fig. 1B). (C) Virus binding competition assay. A total of 1 105 HeLa cells were preincubated with different concentrations of recombinant Ad35 fiber knobs at 4°C for 1 h before incubation with [3H]thymidine-labeled wild-type Ad35 (wtAd35) at a multiplicity of infection of 8,000 VP/cell for another hour, and the number of VP bound per cell was determined after washing with PBS.

Article Snippet: The blot was then incubated with anti-CD46 antibody (clone J4.48; Fitzgerald, Concord, MA) (1:50) in TBS and 3% milk for 1 h at RT and then washed three times for 10 min in TBS-T buffer.

Techniques: Functional Assay, Binding Assay, Mutagenesis, Western Blot, Staining, Incubation, Virus, Competitive Binding Assay, Recombinant, Labeling, Infection

Journal: Journal of Virology

Article Title: Identification of CD46 Binding Sites within the Adenovirus Serotype 35 Fiber Knob

doi: 10.1128/jvi.01732-07

Figure Lengend Snippet: FIG. 4. Structure of the Ad35 fiber knob. (A) Ribbon representation of the Ad35 trimeric fiber knob. Fiber monomers are represented in red, blue, and green, with loop regions between -sheets indicated for the red monomer. (B) Locations of Ad35 fiber amino acids (brown), identified through library analysis, which, upon mutation, decrease binding to CD46 without affecting fiber trimer formation. (C) Representation of two Ad35 trimeric fiber subunits binding to CD46 domains SCR1 and SCR2, based on overlay of the Ad35 crystal structure onto PDB crystal structure 2o39. (D) Representation of two Ad11 trimeric fiber subunits binding to CD46 domains SCR1 and SCR2 from structure 2o39. Differences in proximity between SCR2 and the IJ loops of Ad35 and Ad11 are indicated.

Article Snippet: The blot was then incubated with anti-CD46 antibody (clone J4.48; Fitzgerald, Concord, MA) (1:50) in TBS and 3% milk for 1 h at RT and then washed three times for 10 min in TBS-T buffer.

Techniques: Mutagenesis, Binding Assay

Journal: Journal of Virology

Article Title: Identification of CD46 Binding Sites within the Adenovirus Serotype 35 Fiber Knob

doi: 10.1128/jvi.01732-07

Figure Lengend Snippet: FIG. 5. Location of amino acid residues in the Ad35 fiber knob that are involved in binding to CD46. (A to E) Amino acids in the HI and FG loops, identified through library or model structure analysis, that are involved in HI-FG loop interaction or binding to CD46 domain SCR1. (F to H) Amino acids in the IJ and GH loops, identified through library or structural analysis, that are involved in IJ-GH loop interaction or binding to CD46 domain SCR2. Hydrogen bonds are indicated by dashed yellow lines, and salt bridges are indicated by dashed orange lines.

Article Snippet: The blot was then incubated with anti-CD46 antibody (clone J4.48; Fitzgerald, Concord, MA) (1:50) in TBS and 3% milk for 1 h at RT and then washed three times for 10 min in TBS-T buffer.

Techniques: Binding Assay

Journal: Clinical Medicine Insights. Arthritis and Musculoskeletal Disorders

Article Title: Macroscopical, Histological, and In Vitro Characterization of Nonosteoarthritic Versus Osteoarthritic Hip Joint Cartilage

doi: 10.4137/CMAMD.S29844

Figure Lengend Snippet: Expression of complement receptors (C5aR and C3aR) and CRPs (CD46, CD55, and CD59) in cartilage in situ. FNF and OA cartilage sections immunolabeled for ( A 1–2 ) C5aR, ( B 1–2 ) C3aR, ( C 1–2 ) CD46, ( D 1–2 ) CD55, and ( E 1–2 ) CD59. Notes: Cell nuclei were counterstained using DAPI (blue). Scale bar: 200 µm. Negative controls (B 3 ) performed by using only the secondary antibody revealed no unspecific staining.

Article Snippet: The primary antibodies (rabbit anti-human C3aR [1:30, Assay Biotechnology], mouse anti-human C5aR [1:100, GeneTex, Biozol], mouse anti-human CD46, CD59 [1:20 and 1:40, both AbD Serotec], and goat anti-human CD55 [1:40, R&D Systems]) were diluted in blocking buffer containing 0.1% Triton X-100 (Sigma-Aldrich) and incubated overnight at 4 °C in a humidified chamber.

Techniques: Expressing, In Situ, Immunolabeling, Staining

Journal: Journal of dairy science

Article Title: Yak milk-derived exosomes alleviate lipopolysaccharide-induced intestinal inflammation by inhibiting PI3K/AKT/C3 pathway activation.

doi: 10.3168/jds.2021-20175

Figure Lengend Snippet: Figure 3. Protein–protein interaction network of the CD46 proteins and immune-related proteins and tight junction proteins. Fifteen pro- teins were linked into the network. CDH1 = E-cadherin; OCLN = occluding; TJP1 = ZO-1; LTA = TNF-β. Known interactions are represented:

Article Snippet: The membranes were blocked with 5% skim milk-TBS-Tween20 for 4 h at room temperature and incubated overnight at 4°C with the primary antibodies (diluted 1:1,000 in PBS) against proteins of membrane CD46 (cat. # NB500–301, Novus Biologicals Europe; 1:800), HSP-90α (cat. # 8165, Cell Signaling; 1:1,000), CHI3L1 (cat. # LS-B8213, Life Span BioSciences), APOH (cat. # LS-C314171, Life Span BioSciences, 1:300), tight junction protein 1 (ZO1; cat. # 21773–1-AP, Proteintech; 1:1,000), E-cadherin (cat. # 22018–1-AP, Proteintech; 1:1000), AKT (cat. # 10176–2-AP, Proteintech), phosphorylated (P)-AKT (Ser473; cat. # 4060, Cell Signaling), PI3K (cat. # 2524, Cell Signaling), phosphorylated (P)-PI3K (cat. # 4249T, Cell Signaling), NFκ-B (cat. # 10745–1-AP, Proteintech), C3 (cat. # 21337–1-AP, Proteintech), and β-actin (cat. # abs132184, Absin, Absin Bioscience Co. Ltd.; 1:500).

Techniques:

Journal: Cancer Science

Article Title: Measles virus selectively blind to signaling lymphocyte activity molecule has oncolytic efficacy against nectin‐4‐expressing pancreatic cancer cells

doi: 10.1111/cas.13064

Figure Lengend Snippet: Expression of measles virus receptor molecules on the surface of pancreatic cancer cell lines. (a) Cells were incubated with anti‐nectin‐4 goat polyclonal antibody (gray histogram) or isotype control (black histogram) followed by incubation with Alexa 488‐conjugated rabbit anti‐goat antibody. (b) Nectin‐4 expression is presented as the mean fluorescence intensity (MFI) value by deducting the MFI obtained with isotype control. (c) Cells incubated with anti‐CD46 mouse mAb (red), anti‐signaling lymphocyte activity molecule (SLAM) mouse mAb (blue), or isotype control (black) followed by incubation with Alexa 488‐conjugated goat anti‐mouse antibody.

Article Snippet: The expression of nectin‐4, SLAM, and CD46 was analyzed by flow cytometry according to previously reported methods., The following antibodies were used: anti‐human SLAM mAb (clone 7D4; Biolegend, San Diego, CA, USA); anti‐human CD46 mAb, anti‐nectin‐4 goat polyclonal antibody, mouse control IgG1, and goat control IgG (all from R&D Systems, Minneapolis, MN, USA); and Alexa 488‐conjugated anti‐mouse or anti‐goat antibody (Invitrogen).

Techniques: Expressing, Virus, Incubation, Control, Fluorescence, Activity Assay

Journal: Journal of Virology

Article Title: Neutralizing antibodies with neurotropic factor treatment maintain neurodevelopmental gene expression upon exposure to human cytomegalovirus

doi: 10.1128/jvi.00696-23

Figure Lengend Snippet: NPCs and cerebral organoids express several receptors required for HCMV entry at the plasma membrane. (A) Schematic made in BioRender depicting the HCMV viral particle noting the trimeric (gHgLgO) and pentameric (gHgLpUL128-131A) glycoprotein complexes and target cellular receptors PDGFRα and TGFβRIII (trimer) or Nrp2, THBD, and CD46 (pentamer). (B) RNA expression levels of these entry receptors as determined by analyzing previously performed bulk RNA-seq analysis in cerebral organoids (11). (C) Immunostaining conducted in passage 3 NPCs 3 days post plate down labeling receptors (PDGFRα, Nrp2, and TGFβRII) in green, membrane marker (red), Hoescht (blue), and merged to show the co-localization of membrane and receptor targets. This staining was conducted without permeabilizing the cells to strictly focus on surface expression of the receptors.

Article Snippet: The primary antibodies used were as follows: Nrp2 (R&D Systems, AF2215-SP), PDGFRα (BD Biosciences, 556001), CD46 (R&D Systems, AF2005), THBD (AbCam, ab33513), TGFβRIII (Sigma, T1940), UL123 (Shenk Lab, Clone 1B12), SOX2 (Sigma, AB5603MI), Tuj1 (GeneTex, GTX85469), and nuclear stain Hoescht (Thermo Scientific, H3570), along with a conjugated Cellbrite Orange Cytoplasmic Membrane Dye (Biotium, 30022) in .

Techniques: Clinical Proteomics, Membrane, RNA Expression, RNA Sequencing, Immunostaining, Labeling, Marker, Staining, Expressing