Journal: Cardiovascular Research

Article Title: Heterologous desensitization of cardiac β-adrenergic signal via hormone-induced βAR/arrestin/PDE4 complexes

doi: 10.1093/cvr/cvx036

Figure Lengend Snippet: Heterologous phosphorylation of β2AR promotes β-arrestin 2 dependent recruitment and activation of PDE4D to β2AR. (A) Adult rat ventricular cardiomyocytes were stimulated with different GPCR agonists as indicated (1 µM for 5 min, respectively). Cells were then lysed, and phosphorylation of PDE4D at Serine 190 site was detected in total lysate by western blot using anti-phospho-specific PDE4D antibody. The levels of phosphorylation of PDE4D are quantified and expressed as fold over non-stimulated group (Control). Data are obtained from three independent experiments and shown as mean ± S.E.M. *P < 0.05; ***P < 0.001; ****P < 0.0001 by one-way ANOVA with Dunnett’s multiple comparison test. (B) WT or β-arrestin-2 KO neonatal cardiomyocytes were infected with flag-β2AR together GFP-N-terminal-PDE4D5 adenoviruses. After stimulation with 1 µM PGE2 for 5 min, flag-β2AR was immunoprecipitated using anti-FLAG M2 beads. The elution was subjected to western blot to detect β2AR and PDE4D5, respectively. The PDE4D levels are quantified and expressed as fold over β2AR levels in corresponding non-stimulated groups. (C) Adult rat ventricular cardiomyocytes were infected with mCherry-PDE4D5 together with flag-β2AR or flag-β2AR-PKA4A (lacking four PKA phosphorylation sites) adenovirus and cultured for 48 h. After stimulation with 1 µM PGE2 or 1 µM Angiotensin II (Ang II) for 5 min, cardiomyocytes were lysed and β2AR was immunoprecipitated using anti-FLAG M2 beads. The elution was subjected to western blot to detect β2AR and PDE4D5, respectively. The PDE4D5 levels are quantified and expressed as fold over β2AR levels in corresponding non-stimulated groups. In (B) and (C), data are obtained from four independent experiments and shown as mean ± S.E.M. Between paired groups, n.s, not significant; *P < 0.05 by student’s unpaired t-test.

Article Snippet: Anti-flag antibody (mouse monoclonal IgG2b, Sigma, St. Louis, MO); Phospho-(Ser/Thr) PKA substrate antibody (Rabbit polyclonal IgG, Cell Signaling Technology, Danvers, MA); Phospho-Troponin I (Cardiac) (Ser23/24) antibody (Rabbit polyclonal IgG, Cell Signaling Technology, Danvers, MA); anti-phospho-PDE4D (serine 190) antibody (Rabbit polyclonal IgG, Abcam, Cambridge, United Kingdom); anti-GFP antibody (Rabbit polyclonal IgG, Convance, Princeton, NJ); anti-RFP antibody (Rabbit polyclonal IgG, Rockland, Limerick, PA).

Techniques: Phospho-proteomics, Activation Assay, Western Blot, Control, Comparison, Infection, Immunoprecipitation, Cell Culture

Journal: International journal of oncology

Article Title: Newcastle disease virus induces pro-inflammatory conditions and type I interferon for counter-acting Treg activity.

doi: 10.3892/ijo.2011.1265

Figure Lengend Snippet: Figure 5. Effects of the addition of NDV on the relative expression of IRF-3, IRF-7 and RIG-I transcripts in BM-DCs generated from wt, IRF-3KO, IRF-7KO, IRF-3+IRF-7KO and IFNARKO mice (A) and on the concentration of IFN-α, IFN-β, TNF-α and IL-12 in the corresponding supernatants (B). BM-DC cells derived from the different KO mice were plated in 24-well plates (5x105/well in 24-well plates) and were either treated with PBS or NDV Ulster (30 HU/106 cells). Ten and a half hours later, the cells were harvested in order to allow: (A) the evaluation of IRF-3 (a), IRF-7 (b) and RIG-I (c) transcripts by quantitative real-time PCR and (B) the determination of the concentration of IFN-α (c), IFN-β (d) and TNF-α (e) by ELISA in the corresponding supernatants (B). All protein concentrations are given in pg/ml.

Article Snippet: Mouse IFN-β was measured by ELISA using a rat monoclonal antibody specific for murine IFN-β (United States Biological, MA, USA) and a rabbit polyclonal antibody against murine IFN-β (PBL Interferon Source, NJ, USA).

Techniques: Expressing, Generated, Concentration Assay, Derivative Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: International journal of oncology

Article Title: Newcastle disease virus induces pro-inflammatory conditions and type I interferon for counter-acting Treg activity.

doi: 10.3892/ijo.2011.1265

Figure Lengend Snippet: Figure 2. In vitro production of cytokines and expression of the CD80 marker in human MDDCs upon NDV addition. (A) In vitro induction of the IL-6 and IFN-α protein by Ulster (◆), MTH-68 (▲) strains of NDV and by Sendai virus (◼). Human MDDCs, which were obtained from 2 different donors and gener ated via a 6 days culture in X-vivo™ 15 medium supplemented with GM-CSF and IL-4, were plated in 24-well plates (5x105 cells/well). Four hours later, 3 different viruses were given at various doses (indicated here in HU/106 DC). Supernatants were taken after 24 h and assessed by ELISA for their content in IL-6 [(a) and (c) respectively for donors 1 and 2] and IFN-α [(b) and (d) respec tively for donors 1 and 2]. (B) In vitro production of IFN-α, IL-12 and IL-10 and expression of the CD11c and CD80 markers 10.5 h after co-addition of the Ulster strain of NDV and LPS. Human MDDCs were plated in 24-well plates at 5x105 cells/well. LPS and NDV Ulster were added in triplicates at different concentrations as indicated on the x-axis. The supernatants of each well were collected 24 h later and, after centrifugation, were assessed by ELISA for their contents in IFN-α (a), IL-12 (b) and IL-10 (c). The concentrations of the cyto kines are indicated in pg/ml. The cells were resuspended by incubating the cells with PBS-EDTA 20 mM and analyzed by flow cytometry after double staining with CD11c and CD80 specific mAbs which were coupled to fluorochrome as indicated in Materials and methods. The diagram (d) represents the percentage of CD11c+ CD80+ cells for the different conditions of LPS and NDV tested.

Article Snippet: Mouse IFN-α in supernatants of cultured cells was determined with ELISA using a rat monoclonal antibody and a rabbit polyclonal antibody against murine IFN-α (PBL Interferon Source, NJ, USA).

Techniques: In Vitro, Expressing, Marker, Virus, Enzyme-linked Immunosorbent Assay, Centrifugation, Flow Cytometry, Double Staining

Journal: International journal of oncology

Article Title: Newcastle disease virus induces pro-inflammatory conditions and type I interferon for counter-acting Treg activity.

doi: 10.3892/ijo.2011.1265

Figure Lengend Snippet: Figure 3. Effect of DC maturation and of UV irradiation of NDV on viral transcription (A) and on the type I interferon response induced in mouse BM-DCs (B). (A) Effect of DC maturation and UV irradiation of NDV on viral transcription. Mouse BM-DCs were either matured via a culture for 24 h with LPS (10 µg/ml) or not before being plated at 5x105 cells per well in a 24-well plate. In some tests, NDV was UV-irradiated as described in Materials and methods. Recombinant NDFL-EGFP, which was UV-irradiated or not, was then given (30 HU NDFL-EGFP/106 cells) to each well and the cells were collected 24 h later and evaluated for viral replication by analyzing the EGFP fluorescence by flow cytometry (a) and by determining by quantitative PCR the amount of viral M transcripts (expressed in relative numbers) (b). The EGFP fluorescence is expressed as percentage of EGFP positive cells among the live cells as defined by propidium iodide staining. (B) Effect of DC maturation and UV-irradiation on the induced interferon response. Same conditions as in (A). The interferon response was analyzed by determining the levels of IRF-7 (c) or RIG-I (d) transcripts (expressed in relative numbers) by quantitative PCR (see Materials and methods) and evaluating the amount of IFN-α by ELISA (e). IFN-α is expressed in pg/ml.

Article Snippet: Mouse IFN-α in supernatants of cultured cells was determined with ELISA using a rat monoclonal antibody and a rabbit polyclonal antibody against murine IFN-α (PBL Interferon Source, NJ, USA).

Techniques: Irradiation, Recombinant, Fluorescence, Flow Cytometry, Real-time Polymerase Chain Reaction, Staining, Enzyme-linked Immunosorbent Assay

Journal: International journal of oncology

Article Title: Newcastle disease virus induces pro-inflammatory conditions and type I interferon for counter-acting Treg activity.

doi: 10.3892/ijo.2011.1265

Figure Lengend Snippet: Figure 5. Effects of the addition of NDV on the relative expression of IRF-3, IRF-7 and RIG-I transcripts in BM-DCs generated from wt, IRF-3KO, IRF-7KO, IRF-3+IRF-7KO and IFNARKO mice (A) and on the concentration of IFN-α, IFN-β, TNF-α and IL-12 in the corresponding supernatants (B). BM-DC cells derived from the different KO mice were plated in 24-well plates (5x105/well in 24-well plates) and were either treated with PBS or NDV Ulster (30 HU/106 cells). Ten and a half hours later, the cells were harvested in order to allow: (A) the evaluation of IRF-3 (a), IRF-7 (b) and RIG-I (c) transcripts by quantitative real-time PCR and (B) the determination of the concentration of IFN-α (c), IFN-β (d) and TNF-α (e) by ELISA in the corresponding supernatants (B). All protein concentrations are given in pg/ml.

Article Snippet: Mouse IFN-α in supernatants of cultured cells was determined with ELISA using a rat monoclonal antibody and a rabbit polyclonal antibody against murine IFN-α (PBL Interferon Source, NJ, USA).

Techniques: Expressing, Generated, Concentration Assay, Derivative Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Human molecular genetics

Article Title: Protective effects of mitophagy enhancers against amyloid beta-induced mitochondrial and synaptic toxicities in Alzheimer disease.

doi: 10.1093/hmg/ddab262

Figure Lengend Snippet: Figure 4. Immunoblotting analysis of mitochondrial dynamics proteins. (A) Representative immunoblots for control and mAPP-HT22 cells with or without mitophagy enhancers. (B) Quantitative densitometry analysis for mitochondrial dynamics proteins—significantly increased levels of fission proteins Drp1 and Fis1 were observed in cells transfected with mutant APP. Fusion proteins Mfn1, Mfn2 and Opa1 were significantly decreased. On the other hand, mitophagy enhancers treated mutant APP

Article Snippet: Summary of antibody dilutions and conditions used in the immunoblotting analysis of mitochondrial dynamics, mitochondrial biogenesis, synaptic and mitophagy proteins in mitophagy enhancers treated and untreated mAPP-HT22 cells and untransfected HT22 cells Marker Primary antibody—species and dilution Purchased from company, city and state Secondary antibody, dilution Purchased from company, city and state 6E10 Mouse monoclonal 1:500 Biolegend, San Diego, CA Sheep anti-mouse HRP 1:10000 GE Healthcare Amersham, Piscataway, NJ Drp1 Rabbit polyclonal 1:500 Novus Biological, Littleton, CO Donkey anti-rabbit HRP 1:10000 GE Healthcare Amersham, Piscataway, NJ Fis1 Rabbit polyclonal 1:500 Protein Tech Group, Inc., Chicago, IL Donkey anti-rabbit HRP 1:10000 GE Healthcare Amersham, Piscataway, NJ Mfn1 Rabbit polyclonal 1:400 Abcam, Cambridge, MA Donkey anti-rabbit HRP 1:10000 GE Healthcare Amersham, Piscataway, NJ Mfn2 Rabbit polyclonal 1:400 Abcam, Cambridge, MA Donkey anti-rabbit HRP 1:10000 GE Healthcare Amersham, Piscataway, NJ OPA1 Rabbit polyclonal 1:500 Novus Biological, Littleton, CO Donkey anti-rabbit HRP 1:10000 GE Healthcare Amersham, Piscataway, NJ SYN Rabbit monoclonal 1:400 Abcam, Cambridge, MA Donkey anti-rabbit HRP 1:10000 GE Healthcare Amersham, Piscataway, NJ PSD95 Rabbit monoclonal 1:300 Abcam, Cambridge, MA Donkey anti-rabbit HRP 1:10000 GE Healthcare Amersham, Piscataway, NJ PGC1a Rabbit polyclonal 1:500 Novus Biological, Littleton, CO Donkey anti-rabbit HRP 1:10000 GE Healthcare Amersham, Piscataway, NJ NRF1 Rabbit polyclonal 1:300 Novus Biological, Littleton, CO Donkey anti-rabbit HRP 1:10000 GE Healthcare Amersham, Piscataway, NJ NRF2 Rabbit polyclonal 1:300 Novus Biological, Littleton, CO Donkey anti-rabbit HRP 1:10000 GE Healthcare Amersham, Piscataway, NJ TFAM Rabbit polyclonal 1:300 Novus Biological, Littleton, CO Donkey anti-rabbit HRP 1:10000 GE Healthcare Amersham, Piscataway, NJ PINK1 Rabbit polyclonal 1:500 Novus Biological, Littleton, CO Donkey anti-rabbit HRP 1:10000 GE Healthcare Amersham, Piscataway, NJ Parkin Mouse polyclonal 1:500 Novus Biological, Littleton, CO Sheep anti-mouse HRP 1:10000 GE Healthcare Amersham, Piscataway, NJ

Techniques: Western Blot, Control, Transfection, Mutagenesis