Journal: PLoS ONE

Article Title: Hepatitis C Virus Non-Structural Protein 3 Interacts with Cytosolic 5′(3′)-Deoxyribonucleotidase and Partially Inhibits Its Activity

doi: 10.1371/journal.pone.0068736

Figure Lengend Snippet: (A) HuH7 cells (lanes 1 and 2) or HCV replicon cells (lanes 3-6) were mock-treated (lanes 1, 3 and 5) or treated with interferon-α (10 4 U/ml in lanes 2 and 6; 10 3 U/ml in lane 4). At 72 hrs after treatment, proteins derived from these cells were analyzed using antibodies against NS5A to reflect HCV replication (upper panel), against cdN protein (middle panel) or against Erk-2 as a loading control (bottom panel). (B) The 5′(3′)-deoxyribonucleotidase activity was analyzed using cell lysates derived from (A).

Article Snippet: The primary antibodies used for the analyses in this study were goat anti-cdN polyclonal antibody (Santa Cruz, USA), mouse anti-NS5A monoclonal antibody (Biodesign, USA), mouse anti-myc (4A6) monoclonal antibody (Upstate, USA), mouse anti-V5 monoclonal antibody (Serotec, USA), mouse anti-actin monoclonal antibody (Santa Cruz), mouse anti-NS3 monoclonal antibody (Santa Cruz), mouse anti-mdN monoclonal antibody (Abnova, USA) and rabbit anti-Erk2 polyclonal antibody (Santa Cruz).

Techniques: Derivative Assay, Activity Assay

Journal: PLoS ONE

Article Title: Hepatitis C Virus Non-Structural Protein 3 Interacts with Cytosolic 5′(3′)-Deoxyribonucleotidase and Partially Inhibits Its Activity

doi: 10.1371/journal.pone.0068736

Figure Lengend Snippet: (A) HuH7.5 cells either mock-infected (lane 1) or infected with 2×10 4 HCV infectious particles (lane 2). Three days after infection, proteins derived from these cells were analyzed using antibodies against NS5A (upper panel), cdN (middle panel) or against Erk2 as a loading control (bottom panel). (B) The 5′(3′)-deoxyribonucleotidase activity was analyzed using cell lysates derived from (A).

Article Snippet: The primary antibodies used for the analyses in this study were goat anti-cdN polyclonal antibody (Santa Cruz, USA), mouse anti-NS5A monoclonal antibody (Biodesign, USA), mouse anti-myc (4A6) monoclonal antibody (Upstate, USA), mouse anti-V5 monoclonal antibody (Serotec, USA), mouse anti-actin monoclonal antibody (Santa Cruz), mouse anti-NS3 monoclonal antibody (Santa Cruz), mouse anti-mdN monoclonal antibody (Abnova, USA) and rabbit anti-Erk2 polyclonal antibody (Santa Cruz).

Techniques: Infection, Derivative Assay, Activity Assay

Journal: PLoS ONE

Article Title: Hepatitis C Virus Non-Structural Protein 3 Interacts with Cytosolic 5′(3′)-Deoxyribonucleotidase and Partially Inhibits Its Activity

doi: 10.1371/journal.pone.0068736

Figure Lengend Snippet: (A) HCV replicon cells were transfected with empty vector (lane 2) or the plasmid expressing cdN protein with a V5 tag (lane 3). At 48 hrs after transfection, proteins derived from these cells were analyzed by Western blotting using antibodies against NS5A to reflect HCV replication (upper left panel), against V5 to detect cdN expression (right panel) or against actin as a loading control (bottom left panel). Proteins derived from mock-transfected HuH7 cells (lane 1) were served as a negative control for the detection of NS5A. (B) HCV replicon cells were transduced with lentiviral vectors expressing shLuc (lane 1) or different shRNAs targeting cdN gene (lanes 2-6). After puromycin selection, proteins derived from these cells were analyzed by Western blotting using antibodies against NS5A to reflect HCV replication (upper panel), against cdN protein to determine the knockdown efficiency (middle panel) or against Erk-2 as a loading control (bottom panel).

Article Snippet: The primary antibodies used for the analyses in this study were goat anti-cdN polyclonal antibody (Santa Cruz, USA), mouse anti-NS5A monoclonal antibody (Biodesign, USA), mouse anti-myc (4A6) monoclonal antibody (Upstate, USA), mouse anti-V5 monoclonal antibody (Serotec, USA), mouse anti-actin monoclonal antibody (Santa Cruz), mouse anti-NS3 monoclonal antibody (Santa Cruz), mouse anti-mdN monoclonal antibody (Abnova, USA) and rabbit anti-Erk2 polyclonal antibody (Santa Cruz).

Techniques: Transfection, Plasmid Preparation, Expressing, Derivative Assay, Western Blot, Negative Control, Transduction, Selection

Journal: Journal of virological methods

Article Title: The use of AlphaLISA assay technology to detect interaction between hepatitis C virus-encoded NS5A and cyclophilin A.

doi: 10.1016/j.jviromet.2010.01.020

Figure Lengend Snippet: (A) Schematic representation of the mammalian 2-hybrid system. ‘Protein A’ is expressed as a fusion with the GAL4 DNA-binding domain, and ‘Protein B’ is fused to the VP16 activation domain. Interaction of Proteins ‘A’ and ‘B’ bring the GAL4 and VP16 domains into close proximity, which activate transcription of the luciferase reporter gene located downstream from GAL4 and TATA sequences. (B) Two-hybrid analysis of NS5A/NS5A-CypA interactions in mammalian cells. Reporter plasmid pG5luc was transfected into CHO cells together with plasmid combinations indicated by the ‘x’ axis. Luciferase activity in subsequent cell lysates is expressed as relative light units (RLU) by the ‘y’ axis. Representative data from at least 3 independent repeats are presented as mean RLU +/−standard error of the mean (SEM) calculated from 3 replicates. Open arrows illustrate background levels seen with VP16 fused to NS5A/NS5B; closed arrows highlight RLU generated from VP16-NS5A/NS5B interactions with GAL4-CypA. Mock refers to blank transfection. (C) Indirect immunofluorescence analysis of pACT-NS5A-transfected CHO cells; white arrows indicate co-localization of VP16-NS5A and CypA in the cytoplasm.

Article Snippet: Concomitantly, 5μl of 5mg/ml AlphaLISA Protein G Acceptor beads (Perkin Elmer) were incubated with 15μl of 1mg/ml mouse anti-NS5A monoclonal antibody (Meridian Life Science).

Techniques: Binding Assay, Activation Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Generated, Immunofluorescence

Journal: Journal of virological methods

Article Title: The use of AlphaLISA assay technology to detect interaction between hepatitis C virus-encoded NS5A and cyclophilin A.

doi: 10.1016/j.jviromet.2010.01.020

Figure Lengend Snippet: (A) Alpha assay technology utilizes specific Donor and Acceptor beads and a fluorescence output to reveal protein-protein interactions (see section 3.2). (B) AlphaLISA assessment of the potential to detect interaction between NS5A-His and CypA-FLAG produced by coupled in vitro transcription/translation (IVT/T). Mock refers to blank IVT/T i.e. no DNA added to the reaction. (C) AlphaLISA detection of interaction between purified NS5A-His and GST-CypA WT / GSTCypA H126Q proteins. Reactions were carried out as described in section 2.5.2. Representative data from at least 3 independent repeats are presented as mean RLU +/− SEM calculated from a minimum of 3 replicates.

Article Snippet: Concomitantly, 5μl of 5mg/ml AlphaLISA Protein G Acceptor beads (Perkin Elmer) were incubated with 15μl of 1mg/ml mouse anti-NS5A monoclonal antibody (Meridian Life Science).

Techniques: Fluorescence, Protein-Protein interactions, Produced, In Vitro, Purification

Journal: Journal of virological methods

Article Title: The use of AlphaLISA assay technology to detect interaction between hepatitis C virus-encoded NS5A and cyclophilin A.

doi: 10.1016/j.jviromet.2010.01.020

Figure Lengend Snippet: (A) NS5A-His and GST-CypA WT or GST-CypA H126Q proteins were incubated together with varying concentrations of CsA followed by analysis with specific Alpha Donor/Acceptor beads (see section 2.5.2). Data are presented as mean RLU +/− SEM calculated from a minimum of 3 replicates. Representative data from at least 3 repeats are shown. (B) The IC50 of CsA in the in vitro NS5ACypA AlphaLISA interaction assay was calculated using LabStats add-in for Microsoft Excel and data described in (A).

Article Snippet: Concomitantly, 5μl of 5mg/ml AlphaLISA Protein G Acceptor beads (Perkin Elmer) were incubated with 15μl of 1mg/ml mouse anti-NS5A monoclonal antibody (Meridian Life Science).

Techniques: Incubation, In Vitro

Journal: Journal of virological methods

Article Title: The use of AlphaLISA assay technology to detect interaction between hepatitis C virus-encoded NS5A and cyclophilin A.

doi: 10.1016/j.jviromet.2010.01.020

Figure Lengend Snippet: (A) and (B) Cell lysates prepared from CHO cells transfected with combinations of pcDNA3.1/Hygro(+)-CypAWT-HA, pcDNA3.1/Hygro(+)-CypAH126Q-HA, pCMV-NS5A-His, pCMV-NS5B-His, and pCMV-NS3-His, were assessed for the presence of specific protein complexes using AlphaLISA. CsA was included where indicated and mock refers to blank transfection. Data are presented as mean RLU +/− SEM calculated from a minimum of 3 replicates and representative data from at least 3 repeats are shown. (C) Results from a similar experiment to (B) were used to calculate the IC50 of CsA in the cell-based NS5A-CypA AlphaLISA interaction assay.

Article Snippet: Concomitantly, 5μl of 5mg/ml AlphaLISA Protein G Acceptor beads (Perkin Elmer) were incubated with 15μl of 1mg/ml mouse anti-NS5A monoclonal antibody (Meridian Life Science).

Techniques: Transfection