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: The Journal of Neuroscience

Article Title: Interferon-α Causes Neuronal Dysfunction in Encephalitis

doi: 10.1523/JNEUROSCI.5595-08.2009

Figure Lengend Snippet: Working Memory Correct (WMC) and Repeat Reference Memory (RRM) errors by trial for HIVE and control saline treated mice. HIVE mice that received IFNα NAb made less WMC (p=0.0017) and RRM (p=0.013) errors as memory load increased during trials than HIVE mice that received no treatment or control antibodies (A,B). HIVE mice with IFNα NAb were able to maintain performance similar to control mice without treatment as memory load increased in WMC and RRM errors (C, D). HIVE mice without treatment made significantly more WMC (p=0.016) and RRM (p=0.0002) errors as the memory load increased compared to control and HIVE IFNα NAb mice (C, D).

Article Snippet: Control and HIVE mice received i.p. injections of either 300 μl of saline (control treatment), 100 μg/kg of isotype matched control rabbit polyclonal antibody (control antibody; Vector; Burlingame, CA) or 100 μg/kg of rabbit polyclonal anti-mouse IFNα NAb (PBL, Piscataway, NJ).

Techniques: Control, Saline

Journal: The Journal of Neuroscience

Article Title: Interferon-α Causes Neuronal Dysfunction in Encephalitis

doi: 10.1523/JNEUROSCI.5595-08.2009

Figure Lengend Snippet: Densitometry analysis of microgliosis, astrogliosis, and dendritic arborization in all mice. Densitometry analysis of microgliosis in all control and HIV mouse groups (A). Mice treated with IFNα NAb had significantly less microgliosis compared to other HIVE mouse groups (p=0.0061). Densitometry analysis of astrogliosis in all control and HIV mouse groups (B). There was no statistical difference in astrogliosis between HIV groups. Densitometry analysis of dendritic arborization (MAP2 staining) in all control and HIV mouse groups (C). HIVE mice treated with IFNα NAb did not have a significant loss of dendritic arborization compared to control mice.

Article Snippet: Control and HIVE mice received i.p. injections of either 300 μl of saline (control treatment), 100 μg/kg of isotype matched control rabbit polyclonal antibody (control antibody; Vector; Burlingame, CA) or 100 μg/kg of rabbit polyclonal anti-mouse IFNα NAb (PBL, Piscataway, NJ).

Techniques: Control, Staining

Journal: The Journal of Neuroscience

Article Title: Interferon-α Causes Neuronal Dysfunction in Encephalitis

doi: 10.1523/JNEUROSCI.5595-08.2009

Figure Lengend Snippet: Real Time RT-PCR analysis of IFNα4 and interferon stimulated gene product 15 (ISG15) mRNA in control, HIVE control, HIVE control Ab, and HIVE IFNα NAb mice (A, B). All three treatments of HIV infected mice expressed higher levels of IFNα4 compared to control mice, but there was no significant difference between the three groups. HIV infected mice treated with IFNα NAb produced significantly less ISG15 compared to other HIV infected mice (p=0.0216)(B).

Article Snippet: Control and HIVE mice received i.p. injections of either 300 μl of saline (control treatment), 100 μg/kg of isotype matched control rabbit polyclonal antibody (control antibody; Vector; Burlingame, CA) or 100 μg/kg of rabbit polyclonal anti-mouse IFNα NAb (PBL, Piscataway, NJ).

Techniques: Quantitative RT-PCR, Control, Infection, Produced

Journal: The Journal of Neuroscience

Article Title: Interferon-α Causes Neuronal Dysfunction in Encephalitis

doi: 10.1523/JNEUROSCI.5595-08.2009

Figure Lengend Snippet: Immunohistochemistry staining for PKR in mouse brain tissue. PKR staining in control mouse (A). PKR staining in HIV mouse injected with saline (B). PKR staining in HIV mouse injected with control Ab (C). PKR staining in HIV mouse injected with IFNα NAb (D). Densitometry analysis of PKR staining in untreated control mice and all groups of HIVE mice (E). HIVE mice treated with IFNα NAb had significantly less PKR present in brain tissue compared to other HIVE mice (p<0.0001).

Article Snippet: Control and HIVE mice received i.p. injections of either 300 μl of saline (control treatment), 100 μg/kg of isotype matched control rabbit polyclonal antibody (control antibody; Vector; Burlingame, CA) or 100 μg/kg of rabbit polyclonal anti-mouse IFNα NAb (PBL, Piscataway, NJ).

Techniques: Immunohistochemistry, Staining, Control, Injection, Saline

Journal: The Journal of Neuroscience

Article Title: Interferon-α Causes Neuronal Dysfunction in Encephalitis

doi: 10.1523/JNEUROSCI.5595-08.2009

Figure Lengend Snippet: Immunofluorescence stain of dendritic arbors (MAP2) during a dose response of changes in dendritic arborization to increasing doses IFNα (A). Increasing the dose of IFNα in the neuron culture decreased dendritic arborization of neurons. Dendrite length decreases as the amount of IFNα increases in the neuron cultures (n=100 neurons/dose) (B). 200 IU of IFNα is enough to significantly decreases the length of dendrites (p=0.0003). Dendritic branches decrease as the amount of IFNα increases in the neuron cultures (n=100 neurons/dose) (C). 200 IU of IFNα is enough to decrease the branching of dendrites (p=0.0004). Dendritic length and branching measurements of neuron cultures treated with 300 IU of IFNα and a blocking treatment of IFNα nAb or an NMDA antagonist (MK801 or AP5) (D,E). NMDA antagonists protected against loss of dendritic length (p<0.0001)(D), however, were not able to prevent loss of dendritic branching in the neuron cultures (E).

Article Snippet: Control and HIVE mice received i.p. injections of either 300 μl of saline (control treatment), 100 μg/kg of isotype matched control rabbit polyclonal antibody (control antibody; Vector; Burlingame, CA) or 100 μg/kg of rabbit polyclonal anti-mouse IFNα NAb (PBL, Piscataway, NJ).

Techniques: Immunofluorescence, Staining, Blocking 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-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: 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