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: Cellular Microbiology

Article Title: IFN-γ amplifies NFκB-dependent Neisseria meningitidis invasion of epithelial cells via specific upregulation of CEA-related cell adhesion molecule 1

doi: 10.1111/j.1462-5822.2007.01038.x

Figure Lengend Snippet: Surface expression and de novo transcription of CEACAMs in Chang cells following cytokine stimulation and bacterial infection. Surface expression of CEACAMs on Chang cells was assessed by flow cytometry before and after cytokine stimulation using anti-CEACAM antibody AO115 that binds to multiple CEACAMs. Examples of histograms of CEACAM expression from one experiment are shown (A). CEACAM expression of unstimulated cells is shown in black (filled profile), that of stimulated cells in dotted white lines (traced on to black unfilled profile), and binding of the secondary antibody alone is shown in grey.B and C. Per cent change in the expression of CEACAM and CD46 receptors in response to various cytokines as determined by flow cytometry. AO115 was used to detect CEACAMs (B) and J4-48 for CD46 detection (C) in Chang cells exposed to IFN-γ (diamonds), TNF-α (squares) and IL-1β (triangles) over a 72 h period. Per cent change of MFI observed over untreated cells is shown. Data are means and SEs from three determinations. Note: A and B are separate experiments.D. Agarose gel profile showing the results of a typical semiquantitative RT-PCR of mRNA extracted from Chang cells illustrating the relative levels of 18s rRNA and ceacam1 mRNA present in unstimulated and cytokine-stimulated cells. Lane contents are shown on the right. RT, reverse transcriptase.E. The relative changes in ceacam1 mRNA in cytokine-stimulated Chang cells (24 h) or in cells infected with bacteria (3 h) were calculated after normalizing for 18s rRNA. Means and SEs of 2–4 experiments are shown.F. Western blots showing CEACAM proteins expressed in Chang cells.Top: proteins extracted from unstimulated Chang cells [lane 2 (10 μg), lane 3 (20 μg) and lane 5 (40 μg)] or those exposed to IFN-γ[lane 4 (20 μg) and lane 6 (40 μg)] were analysed by Western blotting using anti-CEACAM antibodies. AO115 binds to multiple CEACAMs but recognized a protein only in stimulated cells, which corresponded to the migration of CEACAM1. Control samples of transfected HeLa cells expressing distinct CEACAMs were used for comparison (lane 1: HeLa-CEA and lane 7: HeLa-CEACAM1; 4 μg total protein of each).Bottom: lanes 1–3: polyclonal AO115 and anti-CEA/CEACAM3 cross-reacting monoclonal antibody COL-1 detection of CEACAMs in HeLa and Chang extracts. Lanes 1 and 4 contain 4 μg each of HeLa-CEA, -CEACAM1 and -CEACAM6. Lanes 2 and 5 contain 60 μg of total protein extract from unstimulated Chang cells, and lanes 3 and 6 contain 60 μg protein from stimulated Chang cells. Data show very low levels of CEACAM1 in unstimulated Chang cells, and only CEACAM1 is upregulated after IFN-γ treatment.

Article Snippet: The mouse monoclonal antibody J4-48 reacts with CD46 (Serotec).

Techniques: Expressing, Infection, Flow Cytometry, Binding Assay, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Bacteria, Western Blot, Migration, Control, Transfection, Comparison

Journal: Cellular Microbiology

Article Title: IFN-γ amplifies NFκB-dependent Neisseria meningitidis invasion of epithelial cells via specific upregulation of CEA-related cell adhesion molecule 1

doi: 10.1111/j.1462-5822.2007.01038.x

Figure Lengend Snippet: CD46 expression in human respiratory cell lines and in Chang conjunctiva cells in response to cytokines.

Article Snippet: The mouse monoclonal antibody J4-48 reacts with CD46 (Serotec).

Techniques: Expressing