Journal: Journal of Virology

Article Title: β-Catenin, a Transcription Factor Activated by Canonical Wnt Signaling, Is Expressed in Sensory Neurons of Calves Latently Infected with Bovine Herpesvirus 1

doi: 10.1128/JVI.02971-15

Figure Lengend Snippet: ORF2 and β-catenin are frequently expressed in the same neuron during latency. Consecutive sections were cut from formalin-fixed paraffin-embedded TG from latently infected calves. (A) For one section, IHC was performed using a peptide-specific ORF2 antibody (1:250 dilution) that has been previously described (16, 22, 42, 43). The adjacent section was stained with the β-catenin antibody (1:200 dilution). Biotinylated goat anti-rabbit IgG (Vector Laboratories) was used as a secondary antibody. Neurons, indicated by numbers 1 to 9, are ORF2+ and β-catenin+ neurons. These results are representative of results from many sections using TG from two calves. (B) For one section, IHC was performed using the β-catenin antibody (1:200 dilution). The consecutive section was stained using a peptide-specific bICP0 antibody (1:250 dilution) that specifically recognizes the bICP0 protein (52, 53). Neurons, indicated by numbers 1 to 12, are β-catenin+ neurons in one section and are also shown in the section stained with the bICP0 antibody to point out the location of the same neurons numbered in the section stained by the β-catenin antibody. These results are representative of four sections cut from TG of two latently infected calves.

Article Snippet: Forty-eight hours after transfection cells were collected and processed for Western blot analysis using a Flag antibody (1:1,000 dilution; Sigma) to detect ORF2, a β-catenin antibody (1:5000 dilution), or an anti-V5 antibody (1:5,000 dilution; Rockland) to detect MAML1.

Techniques: Formalin-fixed Paraffin-Embedded, Infection, Staining, Plasmid Preparation

Journal: Journal of Virology

Article Title: β-Catenin, a Transcription Factor Activated by Canonical Wnt Signaling, Is Expressed in Sensory Neurons of Calves Latently Infected with Bovine Herpesvirus 1

doi: 10.1128/JVI.02971-15

Figure Lengend Snippet: ORF2 and MAML1 stabilized β-catenin and NICD3 protein levels in transfected Neuro-2A cells. (A) Neuro-2A cells were transfected with the designated plasmids (2 μg of DNA for each plasmid). An empty CMV expression vector (pcDNA3.1) was added to maintain equal amounts of plasmid transfected. Forty-eight hours after transfection cells were collected and processed for Western blot analysis using a Flag antibody (1:1,000 dilution; Sigma) to detect ORF2, a β-catenin antibody (1:5000 dilution), or an anti-V5 antibody (1:5,000 dilution; Rockland) to detect MAML1. Fifty micrograms of protein in the total cell lysate was used for each lane. β-Tubulin was used as a loading control. (B) Levels of β-catenin protein in the respective samples presented in panel A were estimated by comparing levels of tubulin to those of β-catenin using a Bio-Rad molecular imager. β-Catenin protein levels in samples transfected with just the β-catenin expression plasmid were normalized to 1, and other values were compared to this result. The results shown in this graph were derived from three independent studies. (C) Neuro-2A cells were transfected with 1 μg of MAML1 expression plasmid alone or with 1 μg to 3 μg of ORF2 as indicated. Forty-eight hours after transfection cells were collected and processed for Western blot analysis using a Flag antibody (1:1,000 dilution; Sigma) or V5 antibody (1:5,000 dilution; Rockland). Fifty micrograms of protein in the total cell lysate was used for each lane, and β-tubulin was used as a loading control. (D) Neuro-2A cells were transfected with DNA constructs (2 μg of DNA for each plasmid) as indicated, and the empty expression vector was added as needed to maintain equal amounts of plasmid. Forty-eight hours after transfection, cells were collected and processed for Western blot analysis using the indicated antibodies at the same concentrations as for the experiments shown in panels A and B. The anti-Notch3 antibody (1:300) (Santa Cruz) was used to detect NICD3. The results shown in panels A, C, and D are representative of four independent experiments. NT, not treated.

Article Snippet: Forty-eight hours after transfection cells were collected and processed for Western blot analysis using a Flag antibody (1:1,000 dilution; Sigma) to detect ORF2, a β-catenin antibody (1:5000 dilution), or an anti-V5 antibody (1:5,000 dilution; Rockland) to detect MAML1.

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Derivative Assay, Construct

Journal: Journal of Virology

Article Title: β-Catenin, a Transcription Factor Activated by Canonical Wnt Signaling, Is Expressed in Sensory Neurons of Calves Latently Infected with Bovine Herpesvirus 1

doi: 10.1128/JVI.02971-15

Figure Lengend Snippet: ORF2 stimulates transactivation potential of MAML1 for β-catenin and NICD-dependent transcription. (A) Neuro-2A cells were cotransfected with 0.1 μg of TOPFlash luciferase reporter, 0.1 μg of β-catenin (S33Y), and 0.1 μg of ORF2. (B) Neuro-2A cells were cotransfected with 0.1 μg of TOPFlash luciferase reporter, 0.1 μg of S33Y (β-catenin), 0.1 μg of MAML1, and increasing concentrations of ORF2 (0.1 to 0.3 μg). (C and D) Neuro-2A cells were cotransfected with 0.3 μg of an HES1 luciferase reporter, 0.3 μg of NICD1 or NICD3, different concentrations of the MAML1 plasmid (0.1 μg and 0.3 μg), and the indicated concentrations of ORF2 (0.1, 0.3, and 1 μg). For all studies, luciferase assays were performed at 48 h after transfection using a dual-luciferase assay. A plasmid expressing Renilla luciferase under the control of a minimal herpesvirus TK promoter was used as an internal control for all samples. The results are the average of three independent experiments, and error bars denote standard errors. An asterisk indicates significant differences (P < 0.05) in promoter activity as determined by one-way analysis of variance and Fisher's least significant difference multiple-means comparison tests.

Article Snippet: Forty-eight hours after transfection cells were collected and processed for Western blot analysis using a Flag antibody (1:1,000 dilution; Sigma) to detect ORF2, a β-catenin antibody (1:5000 dilution), or an anti-V5 antibody (1:5,000 dilution; Rockland) to detect MAML1.

Techniques: Luciferase, Plasmid Preparation, Transfection, Expressing, Control, Activity Assay, Comparison

Journal: Journal of Virology

Article Title: β-Catenin, a Transcription Factor Activated by Canonical Wnt Signaling, Is Expressed in Sensory Neurons of Calves Latently Infected with Bovine Herpesvirus 1

doi: 10.1128/JVI.02971-15

Figure Lengend Snippet: Regulation of cell survival by ORF2, β-catenin, and MAML1 following growth factor withdrawal. (A) Neuro-2A cells were cotransfected with a plasmid expressing β-catenin alone, or with MAML1, a plasmid expressing ORF2, and a plasmid expressing the LacZ gene (transfection control). One microgram of each plasmid was used for transfection, and an empty plasmid was used to maintain equal concentrations of plasmid DNA for the respective transfections. Twenty-four hours after transfection, cells were seeded into new plates at a low density (4,000 cells/cm2) and then incubated with medium containing 0.5% serum for 3 days. Cells were fixed, and β-Gal+ cells were counted as described in Materials and Methods. (B) The number of β-Gal+ Neuro-2A cells transfected with the β-catenin expression plasmid (S33Y) was arbitrarily set at 1. The number of β-Gal+ Neuro-2A cells in the respective studies was normalized to the β-catenin level to calculate the fold of cell survival. The average of four independent experiments is shown. An asterisk denotes significant differences (P < 0.05) in β-Gal+ Neuro-2A cells containing β-catenin with MAML1 plus ORF2 relative to values with other plasmids or combinations, as determined by one-way analysis of variance and Fisher's least significant difference multiple-means comparison tests.

Article Snippet: Forty-eight hours after transfection cells were collected and processed for Western blot analysis using a Flag antibody (1:1,000 dilution; Sigma) to detect ORF2, a β-catenin antibody (1:5000 dilution), or an anti-V5 antibody (1:5,000 dilution; Rockland) to detect MAML1.

Techniques: Plasmid Preparation, Expressing, Transfection, Control, Incubation, Comparison