Journal: PloS one

Article Title: Rabring7 degrades c-Myc through complex formation with MM-1.

doi: 10.1371/journal.pone.0041891

Figure Lengend Snippet: Figure 4. Binding of Rabring7 to c-Myc. A. MBP-Rabring7 or MBP expressed in and purified from E. coli was reacted with 35S-labeled c-Myc, transthyretin (TTR) and MM-1a, and pull-down assays were carried out as described in Experimental procedures. B. HEK293 cells were transfected with various combinations of expression vectors for HA-Rabring7 and FLAG-c-Myc. At 48 hrs after transfection, proteins prepared from cells were immunoprecipitated with an anti-FLAG antibody, and the precipitates were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. C. Proteins prepared from HEK293T cells were immunoprecipitated with an anti-c-Myc antibody or with IgY, and the precipitates were analyzed by Western blotting with anti-Rabring7 and anti-c-Myc antibodies. D. H1299 cells were co-transfected with expression vectors for T7-Rabring7, HA-MM- 1a and FLAG-c-Myc. At 48 hrs after transfection, proteins extracted from cells were immunoprecipitated with an agarose-conjugated anti-FLAG antibody and the proteins bound to agarose bead were eluted with FLAG peptide. The eluted proteins were then immunoprecipitated with an anti- HA antibody and precipitates were analyzed by Western blotting with anti-T7, anti-HA and anti-FLAG antibodies as described in Experimental procedures. doi:10.1371/journal.pone.0041891.g004

Article Snippet: To examine endogenous interaction of c-Myc with Rabring7, proteins extracted from HEK293T cells were immunoprecipitated with an agarose-conjugated chicken anti-c-Myc antibody (Abcam, Cambridge, UK) or with an agarose-conjugated chicken IgY (Acris Antibodies, San Diego, CA) under the same conditions as those for the in vitro binding assay, and the precipitates were subjected to Western blotting with anti-c-Myc (1:1000, N262, Santa Cruz, California, CA) and anti-Rabring7 (1:1000, ab80432, Abcam) antibodies.

Techniques: Binding Assay, Purification, Labeling, Transfection, Expressing, Immunoprecipitation, Western Blot

Journal: PloS one

Article Title: Rabring7 degrades c-Myc through complex formation with MM-1.

doi: 10.1371/journal.pone.0041891

Figure Lengend Snippet: Figure 5. Localization of Rabring7 and MM-1a. A–C. H1299 cells were transfected with various combinations of expression vectors for FLAG- MM-1a, HA-wild-type Rabring7, HA-C229S Rabring7 and FLAG- or EGFP-c-Myc by Lipofectamine 2000. Forty-eight hrs after transfection, the cells were fixed with 4% paraformaldehyde and reacted with anti-FLAG, anti-HA and lamin B antibodies. The cells were then reacted with FITC-conjugated anti- rabbit and rhodamine-conjugated anti-mouse antibodies. A: transfection with FLAG-MM-1a, with FLAG-c-Myc or with HA-wild-type Rabring7 or HA- C229S Rabring7 alone. B: co-transfection with FLAG-MM-1a or FLAG-c-Myc and HA-Rabring7 or HA-C229S Rabring7. C: co-transfection with FLAG- MM-1a, A-wild-type Rabring7 or HA-C229S Rabring7 and EGFP-c-Myc. doi:10.1371/journal.pone.0041891.g005

Article Snippet: To examine endogenous interaction of c-Myc with Rabring7, proteins extracted from HEK293T cells were immunoprecipitated with an agarose-conjugated chicken anti-c-Myc antibody (Abcam, Cambridge, UK) or with an agarose-conjugated chicken IgY (Acris Antibodies, San Diego, CA) under the same conditions as those for the in vitro binding assay, and the precipitates were subjected to Western blotting with anti-c-Myc (1:1000, N262, Santa Cruz, California, CA) and anti-Rabring7 (1:1000, ab80432, Abcam) antibodies.

Techniques: Transfection, Expressing, Cotransfection

Journal:

Article Title: Brain-derived neurotrophic factor (BDNF) contributes to neuronal dysfunction in a model of allergic airway inflammation

doi: 10.1038/sj.bjp.0705638

Figure Lengend Snippet: Animal treatment protocol. Mice were sensitised to OVA adsorbed to Al(OH)3 or vehicle alone by intraperitoneal injections on days 1, 14 and 21. Prior to analysis, animals received two consecutive local aerosol challenges of 1% OVA (w v−1) diluted in PBS or PBS alone and delivered by 20-min aerosolisation on days 26 and 27. Intranasal application of polyclonal chicken IgY (isotype antibody) or anti-mouse BDNF was performed 3 h before each airway allergen challenge. In addition, animals received the antibodies i.p. on day 25. The response to acute allergen exposure was measured on day 35 in the body plethysmograph. All animals were analysed 24 h after the last challenge. Abbreviations: intraperitoneally (i.p.), intranasally (i.n.), aerosol challenge (aerosol).

Article Snippet: Polyclonal chicken IgY anti-mouse BDNF (2 × 25 μ l) (500 μ g ml −1 ) or isotype antibody (500 μ g ml −1 ) (Promega, Madison, U.S.A.) solved in sterile PBS was instilled intranasally 3 h before each airway allergen challenge.

Techniques: Aerosol

Journal:

Article Title: Brain-derived neurotrophic factor (BDNF) contributes to neuronal dysfunction in a model of allergic airway inflammation

doi: 10.1038/sj.bjp.0705638

Figure Lengend Snippet: Overview of experiments and animal numbers

Article Snippet: Polyclonal chicken IgY anti-mouse BDNF (2 × 25 μ l) (500 μ g ml −1 ) or isotype antibody (500 μ g ml −1 ) (Promega, Madison, U.S.A.) solved in sterile PBS was instilled intranasally 3 h before each airway allergen challenge.

Techniques: Aerosol

Journal:

Article Title: Brain-derived neurotrophic factor (BDNF) contributes to neuronal dysfunction in a model of allergic airway inflammation

doi: 10.1038/sj.bjp.0705638

Figure Lengend Snippet: Effect of anti-BDNF treatment on allergen-induced airway obstruction by HBP. Measurement of expiratory flow in response to OVA (intranasal application during continuous measurement of airflow in the HBP). (a) Integration of EF50 values between 15 and 25 min after OVA application. (b) Time course of n=11 animals in each group. Data from three separate experiments are shown. Grey bar: possible artefacts from OVA application. The error bars represent standard error of the mean (s.e.m.). Student́s t-test: ***P<0.001.

Article Snippet: Polyclonal chicken IgY anti-mouse BDNF (2 × 25 μ l) (500 μ g ml −1 ) or isotype antibody (500 μ g ml −1 ) (Promega, Madison, U.S.A.) solved in sterile PBS was instilled intranasally 3 h before each airway allergen challenge.

Techniques:

Journal:

Article Title: Brain-derived neurotrophic factor (BDNF) contributes to neuronal dysfunction in a model of allergic airway inflammation

doi: 10.1038/sj.bjp.0705638

Figure Lengend Snippet: Effect of BDNF and anti-BDNF treatment on inflammation. (a, b) Cytokine levels in BALF. IL-4, IL-5 and IFN-γ were measured in BALF from nonsensitised or OVA-sensitised mice 24 h after aerosol challenge by ELISA. The BAL recovery was 1.4±0.2 ml in all groups. (c, d) Cell differentiation in BALF after OVA aerosol challenge on day 28. Lymphocytes, macrophages, eosinophils and neutrophils were differentiated according to morphological criteria in BALF from nonsensitised or OVA-sensitised mice 24 h after the last aerosol challenge. (e) Cell differentiation in BALF 24 h after nasal OVA challenge on day 36. Student's t-test did not reveal any statistically significant difference (P<0.05) between groups. In all, 12 mice were analysed in each group, in two separate experiments. The error bars represent standard deviation (s.d.).

Article Snippet: Polyclonal chicken IgY anti-mouse BDNF (2 × 25 μ l) (500 μ g ml −1 ) or isotype antibody (500 μ g ml −1 ) (Promega, Madison, U.S.A.) solved in sterile PBS was instilled intranasally 3 h before each airway allergen challenge.

Techniques: Aerosol, Enzyme-linked Immunosorbent Assay, Cell Differentiation, Standard Deviation

Journal:

Article Title: Brain-derived neurotrophic factor (BDNF) contributes to neuronal dysfunction in a model of allergic airway inflammation

doi: 10.1038/sj.bjp.0705638

Figure Lengend Snippet: Effect of BDNF on airway hyper-responsiveness in response to EFS. Airway hyper-responsiveness was measured in response to EFS. The frequency that caused 50% of maximal airway smooth muscle constriction was defined as ES50. Airway contractility was expressed as mean±s.d. Statistics were performed using t-test with Welch's correction. P<0.05 was regarded as a statistically significant difference. (a) Nonsensitised BALB/c mice were treated intranasally either with recombinant human BDNF (5 μg ml−1, 50 μl) or BSA (0.1%, 50 μl) as a control on two consecutive days. AHR was measured 24 h after the last treatment. In all, 12 animals were examined in each group in two separate experiments. (a) OVA-sensitised Balb/c mice were treated intranasally either with anti-BDNF (500 μg ml−1, 50 μl) or istotype IgY (500 μg ml−1, 50 μl) as a control on two consecutive days, 3 h before OVA aerosol challenge. In addition, the animals received 100 μl of the antibody solution i.p. 24 h before the first OVA challenge (day 25). AHR was measured 24 h after the last treatment. A total of 12 animals were examined in each group in two separate experiments.

Article Snippet: Polyclonal chicken IgY anti-mouse BDNF (2 × 25 μ l) (500 μ g ml −1 ) or isotype antibody (500 μ g ml −1 ) (Promega, Madison, U.S.A.) solved in sterile PBS was instilled intranasally 3 h before each airway allergen challenge.

Techniques: Recombinant, Control, Aerosol

Journal:

Article Title: Brain-derived neurotrophic factor (BDNF) contributes to neuronal dysfunction in a model of allergic airway inflammation

doi: 10.1038/sj.bjp.0705638

Figure Lengend Snippet: Effect of anti-BDNF treatment on airway hyper-responsiveness in response to methacholine and capsaicin by HBP. (a) Dose–response curve of expiratory flow (EF50) in response to inhaled methacholine. (b) Dose–response curve of expiratory flow (EF50) in response to inhaled capsaicin. (c) Dose–response curve of sensory irritation (TB) in response to inhaled methacholine. (d) Dose–response curve of sensory irritation (TB) in response to inhaled capsaicin. The error bars represent standard error of the mean (s.e.m.), n=8–10 animals in each group in three separate experiments. Student's t-test: *P<0.05. (a) Nonsensitised against OVA-sensitised+isotype, (d) OVA-sensitised+isotype against OVA-sensitised+anti-BDNF.

Article Snippet: Polyclonal chicken IgY anti-mouse BDNF (2 × 25 μ l) (500 μ g ml −1 ) or isotype antibody (500 μ g ml −1 ) (Promega, Madison, U.S.A.) solved in sterile PBS was instilled intranasally 3 h before each airway allergen challenge.

Techniques:

Journal:

Article Title: Brain-derived neurotrophic factor (BDNF) contributes to neuronal dysfunction in a model of allergic airway inflammation

doi: 10.1038/sj.bjp.0705638

Figure Lengend Snippet: Tachykinin containing sensory neurons from ovalbumin-sensitised mice

Article Snippet: Polyclonal chicken IgY anti-mouse BDNF (2 × 25 μ l) (500 μ g ml −1 ) or isotype antibody (500 μ g ml −1 ) (Promega, Madison, U.S.A.) solved in sterile PBS was instilled intranasally 3 h before each airway allergen challenge.

Techniques: