Journal: Plants

Article Title: Rapid High-Yield Transient Expression of Swine Hepatitis E ORF2 Capsid Proteins in Nicotiana benthamiana Plants and Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope

doi: 10.3390/plants9010029

Figure Lengend Snippet: Transient expression of all constructs. Samples were collected day 2–11 post infiltration and analysed on SDS-PAGE gel ( a ) M. Molecular weight marker (kDa); 1. Leaves inoculated with the empty pEAQ- HT vector; 2–11 Protein samples extracted using TCA method from inoculated plant from day 2 pi to day 11 pi; ( b ) Western blot using anti-HEV ORF2 mAb, the samples are in the same order.

Article Snippet: An E. coli -derived HEV ORF2 recombinant protein (Jena Bioscience, Jena, Germany) of known concentration were used as standards.

Techniques: Expressing, Construct, SDS Page, Molecular Weight, Marker, Plasmid Preparation, Western Blot

Journal: Plants

Article Title: Rapid High-Yield Transient Expression of Swine Hepatitis E ORF2 Capsid Proteins in Nicotiana benthamiana Plants and Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope

doi: 10.3390/plants9010029

Figure Lengend Snippet: ( a ) SDS-PAGE of HEV 110–660; M. Molecular weight marker (kDa); 1. pEAQ- HT empty vector; 2. Crude extract; 3. SN (soluble protein) after extraction in 3× volume extraction buffer and 13k rpm; 4. Pellet (insoluble part); 5. Positive control (HEV ORF2 452–617 aa recombinant protein) ( b ) Western blot with anti-HEV ORF2 mAb, the samples are in the same order; ( c ) Sucrose gradient; M. Molecular weight marker (kDa); 1. SN after pelleting; 2. Dissolved pellet from gradient; 3. 10% sucrose; 4. 20%; 5. 30%; 6. 40%; 7. 50%; 8. 60%; 9. Pellet; 10. Positive control (HEV OF2 452–617 aa recombinant protein).

Article Snippet: An E. coli -derived HEV ORF2 recombinant protein (Jena Bioscience, Jena, Germany) of known concentration were used as standards.

Techniques: SDS Page, Molecular Weight, Marker, Plasmid Preparation, Extraction, Positive Control, Recombinant, Western Blot

Journal: Plants

Article Title: Rapid High-Yield Transient Expression of Swine Hepatitis E ORF2 Capsid Proteins in Nicotiana benthamiana Plants and Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope

doi: 10.3390/plants9010029

Figure Lengend Snippet: ( a ) SDS-PAGE gel and ( b ) Western blot of HEV 110–610 with anti-HEV ORF2 mAb; M. Molecular weight marker (kDa); 1. pEAQ- HT empty vector; 2. Crude extract; 3. SN (soluble protein) after extraction and spin; 4. Pellet-Insoluble part; 5. Positive control (HEV ORF2 452–617 aa recombinant protein); ( c ) SDS-PAGE of sucrose gradient fractions and ( d ) Western blot of fractions using anti-HEV ORF2 mAb; M. Molecular weight marker (kDa); 1 to 12 sucrose fractions from 10% to 60%; 13. Pellet; 14. SN after pelleting; ( e ) Electron microscopy of 30% ( w/v ) fraction and pellet.

Article Snippet: An E. coli -derived HEV ORF2 recombinant protein (Jena Bioscience, Jena, Germany) of known concentration were used as standards.

Techniques: SDS Page, Western Blot, Molecular Weight, Marker, Plasmid Preparation, Extraction, Positive Control, Recombinant, Electron Microscopy

Journal: Plants

Article Title: Rapid High-Yield Transient Expression of Swine Hepatitis E ORF2 Capsid Proteins in Nicotiana benthamiana Plants and Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope

doi: 10.3390/plants9010029

Figure Lengend Snippet: Schematic diagram of the chimeric M2 HEV ORF 2 capsid protein; ( a ) 3D modeling of M2 HEV ORF 2 capsid protein by SWISS MODEL; ( b ) Insertion of 24 amino acid residues of Influenza A virus M2 peptide at the position Gly556 (red) on the P domain of HEV ORF2 capsid protein.

Article Snippet: An E. coli -derived HEV ORF2 recombinant protein (Jena Bioscience, Jena, Germany) of known concentration were used as standards.

Techniques: Virus

Journal: Plants

Article Title: Rapid High-Yield Transient Expression of Swine Hepatitis E ORF2 Capsid Proteins in Nicotiana benthamiana Plants and Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope

doi: 10.3390/plants9010029

Figure Lengend Snippet: ( a ) SDS-PAGE of sucrose gradient of M2 HEV 110–610; M. Molecular weight marker (kDa); 1. TSP; 2. SN after spin the gradient; 3–10. Different sucrose fractions; 11. Pellet; ( b ) Western blot with anti-HEV ORF2 mAb; M. Molecular weight marker (kDa); 1. M2 HEV 110–610 supernatant 2. Fractions four to seven first were pooled and then sedimented by ultracentrifugation; ( c ) TEM of M2 HEV 110–610 after deliberate pelleting.

Article Snippet: An E. coli -derived HEV ORF2 recombinant protein (Jena Bioscience, Jena, Germany) of known concentration were used as standards.

Techniques: SDS Page, Molecular Weight, Marker, Western Blot

Journal: Plants

Article Title: Rapid High-Yield Transient Expression of Swine Hepatitis E ORF2 Capsid Proteins in Nicotiana benthamiana Plants and Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope

doi: 10.3390/plants9010029

Figure Lengend Snippet: ( a ) SDS-PAGE of 3 mg FW; M. Molecular weight marker (kD); 1. Plant inoculated with empty pEAQ- HT ; 2. TSP extracted at two dpi; 3. TSP extracted at four dpi; 4. TSP extracted at six dpi; and ( b ) Western blot with anti-HEV ORF2 mAb of TSP extracted from M2 HEV 110–610 at two dpi (lane 1), at four dpi (lane 2), and at six dpi (lane 3).

Article Snippet: An E. coli -derived HEV ORF2 recombinant protein (Jena Bioscience, Jena, Germany) of known concentration were used as standards.

Techniques: SDS Page, Molecular Weight, Marker, Western Blot

Journal: Journal of Virology

Article Title: Three Amino Acid Mutations (F51L, T59A, and S390L) in the Capsid Protein of the Hepatitis E Virus Collectively Contribute to Virus Attenuation

doi: 10.1128/jvi.02278-10

Figure Lengend Snippet: FIG. 1. Immunofluorescence staining of a subclone of Huh7 cells transfected with similar amounts of capped full-length RNA transcripts. (A) pSHEV-3; (B) mutant rF51L; (C) mutant rT59A; (D) mutant rS390L; (E) mock transfection. Cells were stained for HEV ORF2 protein using chimpanzee 1313 anti-HEV immune serum.

Article Snippet: Briefly, a truncated recombinant ORF2 capsid protein containing the HEV immunodominant region from aa 452 to 617 (GenWay Biotech Inc., San Diego, CA) was used as the antigen.

Techniques: Staining, Transfection, Mutagenesis

Journal: Developmental cell

Article Title: NPC1-mTORC1 signaling Couples Cholesterol Sensing to Organelle Homeostasis and is a Targetable Pathway in Niemann-Pick type C

doi: 10.1016/j.devcel.2020.11.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-CHMP1B , Proteintech , Cat#14639-1-AP; RRID:AB_2079353.

Techniques: Labeling, Plasmid Preparation, Recombinant, Control, Software, Magnetic Beads

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: A Possible Role for Long Interspersed Nuclear Elements-1 (LINE-1) in Huntington’s Disease Progression

doi: 10.12659/MSM.907328

Figure Lengend Snippet: L1 copy content analysis in HD mice. ( A ) Primers used for SYBR Green PCR assay to quantify L1 content. ( B ) 5s-rRNA normalized L1 copy number for 5′UTR, ORF1, and ORF2 in brain caudate regions of wild-type and HD mice (age 10–12 weeks; n=20; includes equal numbers of males and females). Significantly higher L1 copy numbers in HD mice was observed compared with controls. ( C ) L1 content analysis in liver tissues from matched mice used for brain tissue analysis. No change in L1 copy number was observed. ( D ) Age- and sex-based analysis of L1copy number in WT and HD mice at 6 and 16 weeks of age (n=6). No correlation or trend with age or sex was seen. Values are mean ± standard deviation.

Article Snippet: The primary antibodies and the dilutions used were: rabbit LC3B (79D7, Cell Signaling Technology, USA, 1: 1000), rabbit pS6 (D908K, Cell Signaling Technology, USA, 1: 1000), rabbit AMPK alpha (P0056, Sigma, USA 1: 500), rabbit CXCR (UMB2, Abcam USA, 1: 500), rabbit AMP alpha (D6D9, Cell Signaling Technology, USA, 1: 1000), rabbit AKT (C67E7, Cell Signaling Technology, USA, 1: 1000), chicken L1-ORF2 (Rockland Antibodies Inc 1: 500), mouse L1-ORF1 (Millipore, 1: 500) and mouse Actin (8H10D10 Cell Signaling Technology, USA, 1: 2000), and mouse HA (C29F4 Cell Signaling Technology, USA, 1: 2000).

Techniques: SYBR Green Assay, Standard Deviation

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: A Possible Role for Long Interspersed Nuclear Elements-1 (LINE-1) in Huntington’s Disease Progression

doi: 10.12659/MSM.907328

Figure Lengend Snippet: L1 ORFs modulate mTOR activity and AMPKalpha in cells. ( A ) Overexpression of L1-ORF1 and 2 in cells. Immunoblot for HA tag confirming the expression of ORFs in cells. ( B ) mTOR activity in L1-ORF-overexpressed cells. Immunoblot showing decreased mTOR activity in ORF2-overexpressed cells, while no changes were observed in ORF1-overexpressed cells. ( C ) Immunoblot showing decreased AMPK alpha in ORF2-overexpressed cells. ( D ) Quantification of TUNEL-positive cells in ORF1- and ORF2-overexpressed cells. Increased numbers of TUNEL-positive cells were seen in ORF2-overexpressed cells. Values are reported as mean ± standard deviation. ( E ) Immunoblot of LC3B. No changes were seen in LC3B I and II levels in ORF1- and ORF2-overexpressed cells.

Article Snippet: The primary antibodies and the dilutions used were: rabbit LC3B (79D7, Cell Signaling Technology, USA, 1: 1000), rabbit pS6 (D908K, Cell Signaling Technology, USA, 1: 1000), rabbit AMPK alpha (P0056, Sigma, USA 1: 500), rabbit CXCR (UMB2, Abcam USA, 1: 500), rabbit AMP alpha (D6D9, Cell Signaling Technology, USA, 1: 1000), rabbit AKT (C67E7, Cell Signaling Technology, USA, 1: 1000), chicken L1-ORF2 (Rockland Antibodies Inc 1: 500), mouse L1-ORF1 (Millipore, 1: 500) and mouse Actin (8H10D10 Cell Signaling Technology, USA, 1: 2000), and mouse HA (C29F4 Cell Signaling Technology, USA, 1: 2000).

Techniques: Activity Assay, Over Expression, Western Blot, Expressing, TUNEL Assay, Standard Deviation

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: A Possible Role for Long Interspersed Nuclear Elements-1 (LINE-1) in Huntington’s Disease Progression

doi: 10.12659/MSM.907328

Figure Lengend Snippet: L1-ORF2 modulates survival signaling – AKT array. ORF2-overexpressed and control cell lysates were analyzed for changes in survival signaling using AKT array. Protein phosphorylation and total levels were noted as arbitrary absorbance units and their ratio was calculated. Proteins showing greater than the 3-fold decrease in phosphorylation levels were listed along with their site and the potential kinase associated.

Article Snippet: The primary antibodies and the dilutions used were: rabbit LC3B (79D7, Cell Signaling Technology, USA, 1: 1000), rabbit pS6 (D908K, Cell Signaling Technology, USA, 1: 1000), rabbit AMPK alpha (P0056, Sigma, USA 1: 500), rabbit CXCR (UMB2, Abcam USA, 1: 500), rabbit AMP alpha (D6D9, Cell Signaling Technology, USA, 1: 1000), rabbit AKT (C67E7, Cell Signaling Technology, USA, 1: 1000), chicken L1-ORF2 (Rockland Antibodies Inc 1: 500), mouse L1-ORF1 (Millipore, 1: 500) and mouse Actin (8H10D10 Cell Signaling Technology, USA, 1: 2000), and mouse HA (C29F4 Cell Signaling Technology, USA, 1: 2000).

Techniques: Control, Phospho-proteomics, Sequencing

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: A Possible Role for Long Interspersed Nuclear Elements-1 (LINE-1) in Huntington’s Disease Progression

doi: 10.12659/MSM.907328

Figure Lengend Snippet: Immunoblot analysis of specific AKT array proteins from wild-type and HD mouse tissues. Specific AKT target proteins – ASK1, c-Jun, BCL-2, and GSK3beta – were evaluated for phosphorylation changes in brain caudate regions of HD mice. Lanes 1–3: Wild-type mice (n=3); Lanes 4–6: HD mice (n=3). Decreased phosphorylation consistent with the ORF2-overexpression effects were noted in the tissue samples.

Article Snippet: The primary antibodies and the dilutions used were: rabbit LC3B (79D7, Cell Signaling Technology, USA, 1: 1000), rabbit pS6 (D908K, Cell Signaling Technology, USA, 1: 1000), rabbit AMPK alpha (P0056, Sigma, USA 1: 500), rabbit CXCR (UMB2, Abcam USA, 1: 500), rabbit AMP alpha (D6D9, Cell Signaling Technology, USA, 1: 1000), rabbit AKT (C67E7, Cell Signaling Technology, USA, 1: 1000), chicken L1-ORF2 (Rockland Antibodies Inc 1: 500), mouse L1-ORF1 (Millipore, 1: 500) and mouse Actin (8H10D10 Cell Signaling Technology, USA, 1: 2000), and mouse HA (C29F4 Cell Signaling Technology, USA, 1: 2000).

Techniques: Western Blot, Phospho-proteomics, Over Expression

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