Journal: The Journal of general virology

Article Title: Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKbeta-binding protein.

doi: 10.1099/vir.0.020990-0

Figure Lengend Snippet: Fig. 1. Direct association of NIBP with NS5A by GST pull-down assay. LB9.K cells were transiently transfected with FLAG–NIBP. Either GST alone or GST–NS5A purified from E. coli was incubated with cell lysate containing NIBP. Bound proteins were precipitated by glutathione beads as described in Methods and detected by immunoblotting. Both NIBP and NS5A were verified by anti-FLAG (top panel) and anti-NS5A (middle panel) mAbs, showing that GST–NS5A, but not GST alone, associated with FLAG–NIBP. As a control, cell lysates were immunoblotted using anti-GST rabbit polyclonal antibody (bottom panel). IB, Immunoblot. Arrows indicate GST–NS5A and GST alone at approximately 82 and 26 kDa, respectively.

Article Snippet: Rabbit anti-human NIBP polyclonal antibody was from Imgenex.

Techniques: Pull Down Assay, Transfection, Purification, Incubation, Western Blot, Control

Journal: The Journal of general virology

Article Title: Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKbeta-binding protein.

doi: 10.1099/vir.0.020990-0

Figure Lengend Snippet: Fig. 2. NIBP interacts with BVDV NS5A in mammalian cells. (a) c-Myc–NS5A of strain Nose from BVDV and FLAG-tagged NIBP were expressed in LB9.K cells and immunoprecipitated (IP) with anti-c-Myc or anti-FLAG antibody. Immunoprecipitates were subjected to immunoblotting (IB) to detect coprecipitated counterparts. As a negative control, an empty plasmid was used instead of the plasmid encoding FLAG–NIBP or c-Myc–NS5A. Anti-FLAG or anti-c-Myc did not recognize c-Myc-tagged NS5A and FLAG-tagged NIBP, respectively. (b) Endogenous NIBP in LB9.K cells infected with cp and ncp BVDV was immunoprecipitated with normal control rabbit IgG1 (lane 1) or anti-NIBP rabbit IgG (lane 2), and immunoprecipitates were analysed by immunoblotting with specific antibodies.

Article Snippet: Rabbit anti-human NIBP polyclonal antibody was from Imgenex.

Techniques: Immunoprecipitation, Western Blot, Negative Control, Plasmid Preparation, Infection, Control

Journal: The Journal of general virology

Article Title: Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKbeta-binding protein.

doi: 10.1099/vir.0.020990-0

Figure Lengend Snippet: Fig. 3. Determination of the NS5A-binding region in NIBP. (a) Structure and functional domains of NIBP. (b) Full-length or deletion mutants of NIBP used in the study and the results of binding to NS5A. N-terminally FLAG-tagged NIBP mutants encoding the regions indicated were designated 1–1138, 1–786, 624–1138, 191–596 or 528–864, respectively. A summary of immunoprecipitation results is given on the right. (c) Each mutant or full-length NIBP was coexpressed with c-Myc–NS5A in LB9.K cells, immunoprecipitated with an anti-c-Myc antibody and analysed by immunoblotting with an anti-FLAG antibody. As a negative control, an empty plasmid was used instead of the plasmid encoding c-Myc–NS5A. The anti-c-Myc antibody did not recognize FLAG-tagged NIBP or its mutants. The arrow indicates the full-length NIBP from aa 1 to 1138 on the immunoblot.

Article Snippet: Rabbit anti-human NIBP polyclonal antibody was from Imgenex.

Techniques: Binding Assay, Functional Assay, Immunoprecipitation, Mutagenesis, Western Blot, Negative Control, Plasmid Preparation

Journal: The Journal of general virology

Article Title: Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKbeta-binding protein.

doi: 10.1099/vir.0.020990-0

Figure Lengend Snippet: Fig. 4. NS5A colocalizes with NIBP in the ER membrane. LB9.K cells were transiently transfected with FLAG–NIBP expression vector and, 6 h later, were infected with cp or ncp BVDV strains at an m.o.i. of 2 and further incubated for 18 h. The transfected cells were fixed and immunostained with anti-NIBP rabbit polyclonal antibody, anti-Grp 78/Bip or anti-NS5A mAbs. Colocalization was observed by superimposition of green and red images. (a) Anti- NIBP (red) and anti-NS5A (green); (b) anti-NIBP (green) and anti- Grp78/Bip (red).

Article Snippet: Rabbit anti-human NIBP polyclonal antibody was from Imgenex.

Techniques: Membrane, Transfection, Expressing, Plasmid Preparation, Infection, Incubation

Journal: The Journal of general virology

Article Title: Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKbeta-binding protein.

doi: 10.1099/vir.0.020990-0

Figure Lengend Snippet: Fig. 5. NS5A inhibits TNF-a- and poly(I : C)- induced NF-kB activation. Effect of NS5A protein on TNF-a-induced NF-kB activation by luciferase reporter-gene assay. Either HEK293 (a) or LB9.K (b, c) cells were cotransfected with pNF-kB-Luc and pRL-TK reporter plasmids together with empty vectors (pCAGGS or pFLAG–CMV2) or pFLAG– CMV2–NIBP or/and pCAGGS–NS5A expres- sion plasmids, as indicated. At 24 h after transfection, the cells were treated with either TNF-a (10 ng ml”1) for 6 h or poly(I : C) (LB9.K cells only) for 12 h, and NF-kB activity was measured as described in Methods. The results shown are representative of three independent experiments done in duplicate, and values are means±SEM. *P,0.05 (sig- nificantly different from the control; Student’s t-test).

Article Snippet: Rabbit anti-human NIBP polyclonal antibody was from Imgenex.

Techniques: Activation Assay, Luciferase, Reporter Gene Assay, Transfection, Activity Assay, Control

Journal: The Journal of general virology

Article Title: Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKbeta-binding protein.

doi: 10.1099/vir.0.020990-0

Figure Lengend Snippet: Fig. 6. Inhibition of NIBP by siRNA-enhanced viral replication. (a) LB9.K cells (106) were treated with either mock-transfected (–), NIBP-specific (+) or scrambled control non-specific (Scr) siRNAs at a final concentration of 66 nM. In the top panel, an immunoblot probed with anti-NIBP antibody is shown. For the bottom panel, the blot was probed with b-actin internal control. The experiment was repeated more than three times with similar results. (b) At 24 h post-transfection with siRNAs as described above, LB9.K cells were mock infected or infected with cp and ncp BVDV at an m.o.i. of 2. Cells were further incubated for 24 h and BVDV RNA level was determined by real-time PCR analysis. Values are expressed relative to the level of GAPDH RNA. (c) At the same time, the culture supernatants from the cp and ncp BVDV-infected LB9.K cells were collected and used to infect MDBK cells for 50 % tissue culture infective dose (TCID50) assay as described in Methods. The results shown are representative of three independent experiments done in duplicate, and values are means±SEM. *P,0.05 (significantly different from the control; Student’s t-test).

Article Snippet: Rabbit anti-human NIBP polyclonal antibody was from Imgenex.

Techniques: Inhibition, Transfection, Control, Concentration Assay, Western Blot, Infection, Incubation, Real-time Polymerase Chain Reaction, TCID50 Assay

Journal: The Journal of general virology

Article Title: Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKbeta-binding protein.

doi: 10.1099/vir.0.020990-0

Figure Lengend Snippet: Fig. 7. Schematic layout of NIBP–NS5A interaction and the NF- kB pathway. Schematic layout showing a model of BVDV NS5A- mediated TNF-a-induced inhibition of the NF-kB signalling pathway. The inhibition appears to occur at the level of NIK and the IKK complex.

Article Snippet: Rabbit anti-human NIBP polyclonal antibody was from Imgenex.

Techniques: Inhibition

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome

doi: 10.1073/pnas.1018823108

Figure Lengend Snippet: The TRAPPII complex associates with the Rabin8 NH2-terminal domain and is required for Rabin8 centrosomal targeting and primary cilium assembly. (A) LAP-tag purification of Rabin8-associated proteins. (Upper) LAP-Rabin8 and LAP-Rabin81–142 were purified by tandem affinity from 293Trex cells, and the eluted proteins were analyzed by SDS/PAGE (4–12% gradient) and silver stained; then 14 equally spaced gel slices were cut. TRAPPC components had high-percentage peptide coverage in gel slices analyzed by LC-MS/MS corresponding to their predicted molecular weight (Table S1). (Lower) Rabin8-binding domains important for centrosomal vesicular trafficking. (B) TRAPPC9 colocalizes with GFP-Rabin8 on centrosomal vesicles after serum starvation. Micrographs show RPE GFP-Rabin8 cells grown with or without serum for 60 min and fixed and stained with anti-TRAPPC9 antibodies before confocal laser scanning microscopy. (Whole-cell images are shown in Fig. S4E.) (Scale bars: 2 μm.) (C) TRAPPC3, -C9, and -C10 are required for Rabin8 centrosome trafficking. RPE GFP-Rabin8 cells expressing tRFP-Centrin2 were treated with two different ablating TRAPPC protein siRNAs for 72 h (knockdown efficiency is shown in Table S2) followed by serum starvation for 1 h. GFP-Rabin8 subcellular localization was scored in live cells (n = 50) by comparing siRab11a+siRab11b-treated cells (score = 0; Fig. 3C) with siControl-treated cells (score = 5). Results from three independent experiments are shown (Fig. S4H). (D) TRAPPC3, -C9, and -C10 are required for ciliation. RPE cells were treated with siRNAs as in C, serum starved 24 h, and stained with Actub and pericentrin antibodies (n = 75 cells; P < 0.0001). Representative data from three independent experiments are shown.

Article Snippet: Antibodies used were TRAPPC9 (Proteintech), acetylated α-tubulin and γ-tubulin (Sigma), pericentrin (Novus), and GFP ( 3 ).

Techniques: Purification, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Molecular Weight, Binding Assay, Confocal Laser Scanning Microscopy, Expressing