Journal: Virology

Article Title: Receptor usage of a newly emergent adenovirus type 14.

doi: 10.1016/j.virol.2009.02.034

Figure Lengend Snippet: Fig. 2. Competition of viruses for attachment on 293 cells. (A and B) 293 cells were pre-incubated with Ad14-de Wit, Ad14a, Ad3, Ad35 or 10 μg/ml CD46 antibody (MEM-258, Serotec) for 1 h on ice. After washing, 3H-Ad14-de Wit, (A, left panel), 3H-Ad14a, (A, right panel), 3H-Ad3, (B, left panel) or 3H-Ad35 (B, right panel) was added for another hour on ice. After washing, cell-associated radioactivity was determined. N=3. Bars indicate the means. Standard deviation was less than 10% in all cases. (C) Affinity of Ad14-de Wit and Ad14a to 293 cells. To generate Scatchard blots, cells were incubated with increasing MOIs of 3H-Ad14-de Wit or 3H-Ad14a and the number of cells associated viral particles was measured after 1 h of incubation on ice. The y-axis shows the ratio between bound particles to total input particles minus bound particles. The x-axis shows the number of bound particles. The binding affinities (Ka) of virus were calculated on the basis of the slope with standard Excel software as described previously (Tuve et al., 2006).

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: Incubation, Radioactivity, Standard Deviation, Binding Assay, Virus, Software

Journal: Virology

Article Title: Receptor usage of a newly emergent adenovirus type 14.

doi: 10.1016/j.virol.2009.02.034

Figure Lengend Snippet: Fig. 3. Fiber knob competition Ad14-de Wit and Ad14a attachment. (A) Analysis of recombinant Ad fiber knobs. Purified Ad35, Ad14-de Wit, and Ad14a knob proteins (1 μg/lane) were run as native protein (N) or after denaturation (D) on a polyacrylamide gel. Coomassie brilliant blue staining (left panel) revealed the trimeric knob form, which is converted into monomers after boiling. Western blots (right panel) were analyzed for CD46 binding to fiber knobs by subsequent incubation with sCD46, anti-CD46 Mab, and anti-mouse IgG- HRP. (B) Fiber knob competition. Cells were pre-incubated with 0.4 μg or 4 μg knob protein on ice for 1 h. Then 3H-Ad14-de Wit or 3H-Ad14a virus was added and cell-associated radioactivity was measured after 1 hour incubation. N=3. Bars indicate the means. Standard deviation was less than 10% in all cases.

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, Staining, Western Blot, Binding Assay, Incubation, Virus, Radioactivity, Standard Deviation

Journal: Viruses

Article Title: Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization

doi: 10.3390/v13030373

Figure Lengend Snippet: Gene identity, NCBI accession number, and primer sequences of the primers used for the real time PCR.

Article Snippet: The rabbit and mouse monoclonal antibodies against CD55 and CD46 used in flow cytometry experiments, and the recombinant CD55 and CD46 were sourced from Sino Biological Inc. (Beijing, China).

Techniques: Amplification

Journal: Viruses

Article Title: Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization

doi: 10.3390/v13030373

Figure Lengend Snippet: Levels of complement-regulatory protein CD55 remain unaltered, but CD46 levels decline in HeLa cells infected with vesicular stomatitis virus. ( A – G ) Whole cell lysates collected from mock- and VSV-infected HeLa cells at the indicated time points were subjected to immunoblotting to determine the expression of CD46 and CD55. Samples at the time point 0–3 h ( A ), 4–7 h ( B ), 8–11 h ( C ), 12–15 h ( D ), 16–18 h ( E ), 19–21 h ( F ) and 22–24 h ( G ), respectively. Anti-CD55 and CD46 antibodies were used to detect the levels of the corresponding proteins in the lysate at different time points. Virus infectivity was detected using a VSV anti-M antibody while actin served as the loading control. The levels of CD55 were maintained at all of the time points tested compared to the mock; however, the levels of CD46 declined significantly, starting from 15 h onwards (note the decreasing levels of CD46 in D , E , and the complete absence in F , G ). The entire panel of blots is representative of three independent experiments. ( H ) The densitometry analysis of the CD55 and CD46 protein expression, normalized against the loading control. The data represents the mean + SEM of the three independent experiments.

Article Snippet: The rabbit and mouse monoclonal antibodies against CD55 and CD46 used in flow cytometry experiments, and the recombinant CD55 and CD46 were sourced from Sino Biological Inc. (Beijing, China).

Techniques: Infection, Western Blot, Expressing

Journal: Viruses

Article Title: Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization

doi: 10.3390/v13030373

Figure Lengend Snippet: VSV infection causes a decline in the surface expression of CD46 and not CD55. HeLa cells were infected with VSV (10 MOI) for the specified time points. The surface distribution of CD55 and CD46 was determined by staining the mock- and VSV-infected cells with anti-CD55 and CD46 primary antibodies, and by counter staining with AF488-labelled secondary antibody. The two panels, A and B , denote the histogram representing the fluorescence intensity on the x -axis and the cell count on the y -axis. The infection of the HeLa cells with VSV did not alter the surface level expression of CD55 ( A ) even until 24 h; however, a drastic reduction in the surface expression of CD46 ( B ) could be evidenced 6 h post infection.

Article Snippet: The rabbit and mouse monoclonal antibodies against CD55 and CD46 used in flow cytometry experiments, and the recombinant CD55 and CD46 were sourced from Sino Biological Inc. (Beijing, China).

Techniques: Infection, Expressing, Staining, Fluorescence, Cell Counting

Journal: Viruses

Article Title: Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization

doi: 10.3390/v13030373

Figure Lengend Snippet: Vesicular stomatitis virus infection leads to down-regulation of CD55 and CD46 transcripts. A comparative analysis of the relative levels of CD55 ( A ) and CD46 ( B ) mRNA in VSV and mock-infected HeLa cells at 6, 12, 18 and 24 h was carried out by RT-qPCR. The total RNA isolated from the mock- and VSV-infected cells was converted to cDNA. Equal concentrations of cDNA from all of the samples were used to analyze the gene expression at various time points post VSV-infection using Taqman gene expression assays. The fold change was calculated by the comparative Ct method (2^- ddCt). The statistical significance was calculated using Students t -test, with * p ≤ 0.01; ** p ≤ 0.001; **** p ≤ 0.0001, and ns = non-significance.

Article Snippet: The rabbit and mouse monoclonal antibodies against CD55 and CD46 used in flow cytometry experiments, and the recombinant CD55 and CD46 were sourced from Sino Biological Inc. (Beijing, China).

Techniques: Infection, Quantitative RT-PCR, Isolation, Expressing

Journal: Viruses

Article Title: Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization

doi: 10.3390/v13030373

Figure Lengend Snippet: Cycloheximide chase assay to measure protein stability. ( A ) The HeLa cells were treated with Cycloheximide for the indicated times, and the whole cell lysate was subjected to immunoblotting in order to determine the stability of CD55 and CD46. β-actin was used as the loading control, and P53 served as the positive control. ( B ) Quantification of the immunoblot results by Image J software. The result represented is the average + SEM of four independent experiments obtained by normalizing the band intensity of the RCA against b-actin. The statistical significance was calculated using Student’s t -test.

Article Snippet: The rabbit and mouse monoclonal antibodies against CD55 and CD46 used in flow cytometry experiments, and the recombinant CD55 and CD46 were sourced from Sino Biological Inc. (Beijing, China).

Techniques: Western Blot, Positive Control, Software

Journal: Viruses

Article Title: Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization

doi: 10.3390/v13030373

Figure Lengend Snippet: Complement regulator CD55 is found in greater abundance than CD46 on VSV from HeLa cells. ( A ) Equal concentrations of protein in the purified virus (5 μg) were separated by SDS-PAGE and subjected to Western blotting. The proteins that have been probed are indicated in the right, and their corresponding molecular weights are in parentheses. ( B ) An ELISA specific to VSV was performed by coating wells with serially-diluted gradient-purified VSV. The adsorbed virus particles were detected using an anti-VSV-G antibody. Variability in the absorbance was observed even at similar concentration of viruses purified at varying time intervals. Across the samples, an absorbance of ~1.3 was found to be common; this is indicated by the lines drawn against the optical density. ( y -axis) to the corresponding concentrations ( x -axis).

Article Snippet: The rabbit and mouse monoclonal antibodies against CD55 and CD46 used in flow cytometry experiments, and the recombinant CD55 and CD46 were sourced from Sino Biological Inc. (Beijing, China).

Techniques: Purification, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Viruses

Article Title: Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization

doi: 10.3390/v13030373

Figure Lengend Snippet: Levels of CD55 and CD46 associated with sucrose gradient-purified VSV from HeLa cells at various time intervals. a,b The concentration of the virion-associated CD55/CD46 was calculated from the pixel intensity of the known concentrations of rCD46 and rCD55 run in parallel ( Figure 5 A). The concentration of CD55 and CD46 is depicted as ng/5μg of VSV. c,f Based on the densitometric analysis of VSV-G or M, further normalization was performed relative to the values obtained at 6–12 h (taken as 1). The values indicate the decline in the concentration of G or M relative to 6–12 h. d,g The ratio of CD55 to VSV-G or VSV-M was calculated by dividing the CD55 concentration at a specific time point by the normalized levels of G or M. The values in d were obtained by dividing a by c , and those in e by dividing b by c . Similarly, the ratios in g and h were obtained by dividing a by f and b by f , respectively.

Article Snippet: The rabbit and mouse monoclonal antibodies against CD55 and CD46 used in flow cytometry experiments, and the recombinant CD55 and CD46 were sourced from Sino Biological Inc. (Beijing, China).

Techniques: Concentration Assay

Journal: Viruses

Article Title: Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization

doi: 10.3390/v13030373

Figure Lengend Snippet: CD55 confers greater resistance to VSV against complement compared to CD46. ( A ) Western blot depicting the level of expression of CD55 and CD46 in HeLa and A549 cells; β-actin served as the equal loading control. ( B ) The effect of NHS in neutralizing VSV grown in HeLa and A549 cells was assessed by a plaque reduction assay. The virus harvested at the indicated time intervals was incubated either with NHS or PBS (black bars). At all of the time points tested, the HeLa-grown viruses showed marked resistance to complement-mediated neutralization. The degree of neutralization of A549-grown VSV known to harbor less CD55 was significantly higher than that of HeLa-grown VSV at 12–18 h ( p < 0.0001). The symbols in the graph represent * p < 0.05; ** p < 0.005; *** p < 0.0005). The additional symbols represent the comparison of significance between A549- and HeLa-grown VSV treated with NHS at the respective time ranges, where # p < 0.0005; $ p < 0.0005; ^ p < 0.0001; @ p < 0.005.

Article Snippet: The rabbit and mouse monoclonal antibodies against CD55 and CD46 used in flow cytometry experiments, and the recombinant CD55 and CD46 were sourced from Sino Biological Inc. (Beijing, China).

Techniques: Western Blot, Expressing, Incubation, Neutralization

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 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: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: The classical pathway is triggered by interaction of C1 with immune or non-immune complexes leading to conformational changes in C1q, activation of C1r and C1s, and subsequent assembly of C3 convertase. The lectin pathway is activated by binding of mannose-binding lectin (MBL) to mannose residues on the pathogen surface, which activates the MBL-associated serine proteases (MASPs), followed by formation of C3 convertase. Spontaneous hydrolysis of C3 initiates the alternative complement pathway. All three pathways of the complement cascade converge on the classical C3 convertase, which cleaves and activates component C3, forming C3a and C3b. This triggers a series of further cleavage and activation events, leading to cleavage of C5 into C5a and C5b, and eventual formation of the membrane attack complex (MAC), consisting of C5b, C6, C7, C8, and C9. MAC is the terminal cytolytic complex of the complement pathway; it causes osmotic lysis of target cells by forming transmembrane channels that disrupt the phospholipid bilayer of target cells. The complement system is tightly regulated by the following complement control proteins: CFH, CFHR1, CFHR4, CFB, CD55, CD59, CD46, CFI, and CFP.

Article Snippet: 7 , CD46 , 44 kDa , Goat Anti-CD46 Polyclonal antibody # AF2005 (RD Systems) , 1:1000 , Human , Dnk pAb to Goat IgG (HRP) ab97120 (ABCAM) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Activation Assay, Binding Assay, Lysis

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Complement Markers’ Information.

Article Snippet: 7 , CD46 , 44 kDa , Goat Anti-CD46 Polyclonal antibody # AF2005 (RD Systems) , 1:1000 , Human , Dnk pAb to Goat IgG (HRP) ab97120 (ABCAM) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Marker, Activation Assay, Lysis, Inhibition

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Complement Genes’ Information.

Article Snippet: 7 , CD46 , 44 kDa , Goat Anti-CD46 Polyclonal antibody # AF2005 (RD Systems) , 1:1000 , Human , Dnk pAb to Goat IgG (HRP) ab97120 (ABCAM) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Variant Assay

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Complement Proteins’ Western Blotting Information.

Article Snippet: 7 , CD46 , 44 kDa , Goat Anti-CD46 Polyclonal antibody # AF2005 (RD Systems) , 1:1000 , Human , Dnk pAb to Goat IgG (HRP) ab97120 (ABCAM) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Complement Gene Expression Data.

Article Snippet: 7 , CD46 , 44 kDa , Goat Anti-CD46 Polyclonal antibody # AF2005 (RD Systems) , 1:1000 , Human , Dnk pAb to Goat IgG (HRP) ab97120 (ABCAM) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Decreased protein levels of CFH, CFHL1, CD55, CD59, CFI, and CD46, and increased levels of CFP, CFB, CFHR4, and CFHR1 protein in AMD cybrids. (A, C, E, G, I, K, M, O, Q, S) Representative Western blots of CFH, CFHL1, CD55, CD59, CFI, CFP, CFB, CD46, CFHR4, and CFHR1 respectively. (B, D, F, H, J, L, N, P, R, T) Graphs showing quantitation of CFH, CFHL1, CD55, CD59, CFI, CFP, CFB, CD46, CFHR4, and CFHR1 proteins in Older-Normal and AMD cybrids. * P < 0.05, ** P < 0.01. n = 4–5. Data were analyzed using Student’s T-test.

Article Snippet: 7 , CD46 , 44 kDa , Goat Anti-CD46 Polyclonal antibody # AF2005 (RD Systems) , 1:1000 , Human , Dnk pAb to Goat IgG (HRP) ab97120 (ABCAM) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Western Blot, Quantitation Assay

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Complement Protein Expression Data.

Article Snippet: 7 , CD46 , 44 kDa , Goat Anti-CD46 Polyclonal antibody # AF2005 (RD Systems) , 1:1000 , Human , Dnk pAb to Goat IgG (HRP) ab97120 (ABCAM) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Expressing