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: Cancer Immunology Research

Article Title: MicroRNAs Affect Complement Regulator Expression and Mitochondrial Activity to Modulate Cell Resistance to Complement-Dependent Cytotoxicity

doi: 10.1158/2326-6066.cir-18-0818

Figure Lengend Snippet: Figure 6. Modulation of the membrane complement regulatory proteins by microRNAs. The expression of CD46 (A), CD55 (B), and CD59 (C) in lysates of K562 cells transfected with microRNA inhibitors or with control, determined by the proteomic analysis described in Materials and Methods, is shown as the mean SD of log2 of intensity value units of five independent experiments. , P < 0.05; , P < 0.01, relative to control (Student t test). NS, not significant. D, K562 cells were transfected with miR-616 inhibitor plasmid or with control (C) plasmid as a negative control. After 48 hours, the cells were labeled with anti-CD46, anti-CD55, or anti-CD59 and then with fluorescently labeled secondary antibody. Cells were then analyzed by flow cytometry, and the mean fluorescence intensity (MFI) values, representative of three independent experiments, were determined. The expression of each regulator in anti–miR-616–treated cells was normalized to their levels in control cells (set as 100). , P < 0.05; , P < 0.01, relative to control (Student t test). E–G, K562 cells were transfected with a miR-150 expression plasmid or a control plasmid. After 24 hours, the cells were labeled with mouse anti-CD46 (E), anti-CD55 (F), or anti-CD59 (G) and fluorescently labeled secondary antibody. Cells were then analyzed by flow cytometry, and MFI values, representative of three independent experiments, were determined. , P < 0.05, relative to control (Student t test).

Article Snippet: Mouse monoclonal antihuman CD46 (clone MEM-258), anti-human CD55 (clone 67), and anti-human CD59 (clone MEM-43) antibodies were purchased from AbD Serotec.

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

Journal: Gene therapy

Article Title: Measles virus selectively blind to signaling lymphocyte activation molecule as a novel oncolytic virus for breast cancer treatment.

doi: 10.1038/gt.2012.44

Figure Lengend Snippet: Figure 2. PVRL4 and not CD46 is involved in the infection of breast cancer cell lines with rMV-SLAMblind. (a) Surface expression of CD46 and PVRL4 in breast cancer cell lines analyzed by flow cytometry. (Left) Cells incubated with anti-CD46 mouse MAb (gray histogram) or isotype control (white histogram) followed by incubation with Alexa-488-conjugated goat anti-mouse antibody. (Right) Cells incubated with anti- PVRL4 goat polyclonal antibody (gray histogram) or isotype control (white histogram) followed by incubation with Alexa-488-conjugated rabbit anti-goat antibody. (b) Cells were pretreated with anti-CD46 or anti-PVRL4 antibody and infected with rMV-EGFP, rMV-EGFP-SLAMblind or rMV-Edmonston at an MOI of 0.1. Cells were incubated in medium with or without antibody and were photographed at 2 (Vero) or 3 d.p.i. (the others). Cells infected with rMV-Edmonston were immunostained using anti-N rabbit polyclonal antibody. Magnification: 100. (c) CHO- K1 cells were transfected with pCAG-hCD46 or the empty vector (pCAGGS). After 2 days, they were infected with rMV, rMV-SLAMblind or rMV- Edmonston at an MOI of 1 and incubated in the presence of fusion inhibitory peptide. At 2 d.p.i., cells were fixed and immunostained using anti-N MAb and the number of infected cells was counted. Infectivity in cells transfected with pCAGGS was set as 100%. Error bars indicate s.e. *Po0.001 versus pCAGGS. (d) BHK cells were transfected with pCAG-hPVRL4 or pCAGGS. After two days, they were infected with rMV, rMV- SLAMblind or rMV-Edmonston at an MOI of 0.1 and incubated in the presence of fusion inhibitory peptide. At 2 d.p.i., infectivity was determined as described in (c). *Po0.001 versus pCAGGS.

Article Snippet: The following antibodies were used as primary antibodies: anti-human SLAM MAb (clone 7D4; Biolegend, San Diego, CA, USA), anti-human CD46 MAb, anti-PVRL4 goat polyclonal antibody (R&D Systems), mouse control IgG1 (R&D Systems) and goat control IgG (R&D Systems).

Techniques: Infection, Expressing, Cytometry, Incubation, Control, Transfection, Plasmid Preparation

Journal: Gene therapy

Article Title: Measles virus selectively blind to signaling lymphocyte activation molecule as a novel oncolytic virus for breast cancer treatment.

doi: 10.1038/gt.2012.44

Figure Lengend Snippet: Figure 3. Infection of CD46-positive normal human cells. (a) Surface expression of CD46, PVRL4 and SLAM in NHDFs analyzed by flow cytometry as described in Figures 1c and 2a. (b, c) NHDFs were infected with rMV-SLAMblind or rMV-Edmonston at an MOI of 1. (b) Cells fixed and immunostained with anti-N MAb at 3 d.p.i. Magnification: 100. (c) Cell viability measured at each time point by WST-1 assay.

Article Snippet: The following antibodies were used as primary antibodies: anti-human SLAM MAb (clone 7D4; Biolegend, San Diego, CA, USA), anti-human CD46 MAb, anti-PVRL4 goat polyclonal antibody (R&D Systems), mouse control IgG1 (R&D Systems) and goat control IgG (R&D Systems).

Techniques: Infection, Expressing, Cytometry, WST-1 Assay

Journal: Molecular Medicine Reports

Article Title: The effect of anti-HLA class I antibodies on the immunological properties of human glomerular endothelial cells and their modification by mTOR inhibition or GCN2 kinase activation

doi: 10.3892/mmr.2021.11994

Figure Lengend Snippet: Effect of anti-HLAI on ICAM-1, HLA-DR, CD46 and CD59, and the impact of halofuginone or everolimus treatment. (A) Representative experiment for each of the evaluated factors. (B) Cumulative results are presented. Anti-HLAI antibodies upregulated ICAM-1, HLA-DR, CD46 and CD59. Halofuginone or everolimus treatment decreased ICAM-1. Data are presented as the mean ± SEM. *P<0.05 vs. control cells, # P<0.05 vs. anti-HLAI-treated cells, ^ P<0.05 vs. anti-HLAI-treated cells administered halofuginone, + P<0.05 vs. anti-HLAI-treated cells administered everolimus and $ P<0.05 vs. anti-HLAI-treated cells with halofuginone and everolimus. HLAI, human leukocyte antigen class I; ICAM-1, intracellular adhesion molecule-1; Hal, halofuginone; Ever, everolimus; Ctrl, control.

Article Snippet: Blots were incubated at 4°C for 16 h with the primary antibodies specific against activated cleaved caspase-3 (cleaved caspase-3, 1:1,000, cat. no ab13847, Abcam), focal adhesion kinase (FAK, 1:100, cat. no sc-271126, Santa Cruz Biotechnology, Inc.), phosphorylated at Tyr397 FAK (p-FAK, 1:1,000, cat. no 8556, Cell Signaling Technology, Inc.), mTOR (1:100, cat. no sc-517464, Santa Cruz Biotechnology, Inc.), phosphorylated at Ser2448 mTOR (p-mTOR, 1:100, cat. no sc-293133, Santa Cruz Biotechnology, Inc.), p70S6 kinase (p70S6K, 1:100, cat. no sc-8418, Santa Cruz Biotechnology, Inc.), phosphorylated at Thr389 p70S6K (p-p70S6K, 1:1,000, cat. no 9234, Cell Signaling Technology), protein kinase B (Akt, 1:100, cat. no sc-5298, Santa Cruz Biotechnology, Inc.), phosphorylated at Ser474 Akt (p-Akt, 1:1,000, cat. no 4060, Cell Signaling Technology, Inc.), GCN2 kinase (GCN2K, 1:100, cat. no sc-374609, Santa Cruz Biotechnology, Inc.), phosphorylated at Thr899 GCN2K (p-GCN2K, 1:1,000, cat. no ab75836; Abcam), eIF2α (1:100, cat. no sc-133132, Cell Signaling Technology, Inc.), phosphorylated at Ser51 eIF2α (p-eIF2a, 1:1,000, cat. no 9721, Cell Signaling Technology, Inc.), intercellular adhesion molecule 1 (ICAM-1, 1:1,000, cat. no 4915; Cell Signaling Technology), HLA-DR (Ultra-LEAFTM Purified anti-human HLA-DR Antibody, cat. no 307648, Biolegend), CD46 (1:1,000, cat. no CSB-PA923298, Cusabio), CD59 (1:1,000, cat. no CSB-PA004947YA01HU, Cusabio), and β-actin (1:5,000, cat no. 4967, Cell Signaling Technology, Inc.).

Techniques: Control