Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Elevated bone marrow sympathetic drive precedes systemic inflammation in angiotensin II hypertension

doi: 10.1152/ajpheart.00510.2018

Figure Lengend Snippet: Antibodies used for flow cytometry and brain immunohistochemistry analysis in rat

Article Snippet: Data were normalized to GAPDH and presented as means ± SE. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Assay ID Abbreviated Name Full Name Vendor Type Rn00562488_s1 Adra2a Adrenoceptor-α2A ThermoFisher TaqMan Rn00593312_s1 Adra2b Adrenoceptor-α2B ThermoFisher TaqMan Rn00593341_s1 Adra2c Adrenoceptor-α2C ThermoFisher TaqMan Rn00824536_s1 Adrb1 Adrenoceptor-β1 ThermoFisher TaqMan Rn00560650_s1 Adrb2 Adrenoceptor-β2 ThermoFisher TaqMan Rn00580555_m1 Ccl2 chemokine (C-C motif) ligand 2 ThermoFisher TaqMan Rn01637698_s1 Ccr2 chemokine (C-C motif) receptor 2 ThermoFisher TaqMan Rn01472831_m1 Hifla hypoxia-inducible factor 1, α-subunit ThermoFisher TaqMan Rn00709342_m1 Itgam integrin, α-M ThermoFisher TaqMan Rn00569848_m1 Tlr4 Toll-like receptor 4 ThermoFisher TaqMan Open in a separate window Real-time PCR primers in the rat

Techniques: Flow Cytometry, Immunohistochemistry

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Elevated bone marrow sympathetic drive precedes systemic inflammation in angiotensin II hypertension

doi: 10.1152/ajpheart.00510.2018

Figure Lengend Snippet: Real-time PCR primers in the rat

Article Snippet: Data were normalized to GAPDH and presented as means ± SE. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Assay ID Abbreviated Name Full Name Vendor Type Rn00562488_s1 Adra2a Adrenoceptor-α2A ThermoFisher TaqMan Rn00593312_s1 Adra2b Adrenoceptor-α2B ThermoFisher TaqMan Rn00593341_s1 Adra2c Adrenoceptor-α2C ThermoFisher TaqMan Rn00824536_s1 Adrb1 Adrenoceptor-β1 ThermoFisher TaqMan Rn00560650_s1 Adrb2 Adrenoceptor-β2 ThermoFisher TaqMan Rn00580555_m1 Ccl2 chemokine (C-C motif) ligand 2 ThermoFisher TaqMan Rn01637698_s1 Ccr2 chemokine (C-C motif) receptor 2 ThermoFisher TaqMan Rn01472831_m1 Hifla hypoxia-inducible factor 1, α-subunit ThermoFisher TaqMan Rn00709342_m1 Itgam integrin, α-M ThermoFisher TaqMan Rn00569848_m1 Tlr4 Toll-like receptor 4 ThermoFisher TaqMan Open in a separate window Real-time PCR primers in the rat

Techniques: Real-time Polymerase Chain Reaction

Journal: Journal of Veterinary Science

Article Title: In vitro effects of meloxicam on the number, Foxp3 expression, production of selected cytokines, and apoptosis of bovine CD25+CD4+ and CD25-CD4+ cells

doi: 10.4142/jvs.2013.14.2.125

Figure Lengend Snippet: The effect of MEL on IL-10 (A), TGF-β (B), and IFN-γ (C) production. The results are expressed as a percentage of CD25 high CD4+, CD25 low CD4+, and CD25-CD4+ cells expressing IL-10, TGF-β, or IFN-γ. Typical cytograms (D) illustrating IFN-γ expression in particular lymphocyte subpopulations are shown. Results are presented as the mean ± SE (n = 18) of three independent experiments (each one using six animals). * p < 0.01, MEL-treated cells versus the control cells.

Article Snippet: After extracellular staining (as described above) and fixation (200 μL 2% paraformaldehyde in Dulbecco's PBS per sample for 15 min on ice; both reagents were purchased from Sigma-Aldrich, USA), the cells were permeabilized with 2 mL SAP buffer [0.1% (w/v) saponin and 0.05% (w/v) NaN 3 in Hanks' balanced salt solution (HBSS; all from Sigma-Aldrich, USA)] and stained with APC (allophycocyanin) -conjugated mouse anti-TGF-β mAb (1 : 20, 1D11, IgG1, this antibody reacts with human, mouse, and bovine TGF-β1 and TGF-β2; R&D Systems, USA) for 45 min at RT in the dark.

Techniques: Expressing, Control

Journal: Hypertension (Dallas, Tex. : 1979)

Article Title: Type III Transforming Growth Factor-β Receptor Drives Cardiac Hypertrophy Through β-Arrestin2-Dependent Activation of Calmodulin-Dependent Protein Kinase II.

doi: 10.1161/HYPERTENSIONAHA.116.07420

Figure Lengend Snippet: Figure 1. Type III transforming growth factor-β receptor (TβRIII) expression increases during hypertrophic development. A, Relative mRNA expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), β-myosin heavy chain (β-MHC), and TβRIII in human hypertrophic heart samples (CH) and human nonhypertrophic heart samples (NCH). B, Representative Western blotting demonstrating expression profile of ANP, β-MHC, and TβRIII protein in CH and NCH. C, Summarized relative protein expression from the experiments shown in B (CH, n=8; NCH, n=6; *P<0.05 vs NCH). D, Representative Western blotting (a) and summarized TβRIII expression data (b) in left ventricle tissue (LVT) of the mice administrated with isoproterenol (ISO) infusion for 3, 7, 14, 21, and 28 d, respectively (n=7/group; *P<0.05 vs Ctrl). E, Representative Western blotting (a) and summarized TβRIII expression data (b) in LVT 1, 2, 3, and 4 wk post transverse aortic constriction (TAC; n=7/group; *P<0.05 vs Sham). F, Representative immunofluorescence staining images of TβRIII, α-actinin, nuclei, and their overlay in mouse hearts (a) and representative histograms of fluorescence intensity (b) constructed across myocardium (the yellow solid line in a shows where the data were taken). All data presented as mean±SD.

Article Snippet: The primary antibodies used in the present study included anti-TβRIII antibody (1:1000 dilution, Cell signaling #2519; 1:200 dilution, Santa Cruz, sc-28975, sc-6199), anti-β-arrestin2 antibody (1:1000 dilution, Cell Signaling #3857; 1:200 dilution, Santa Cruz, sc-13140), anti-p-CaMKII antibody (1:1000 dilution, Cell Signaling, #3361), anti-CaMKII antibody (1:200 dilution, Santa Cruz, sc-13082), anti-ANP antibody (1: 200 dilution, Santa Cruz, sc-20158), anti-β-MHC (1:5000 dilution, Sigma-Aldrich, M8421), anti-β1-AR antibody (1: 200 dilution, Santa Cruz, sc-568), anti-β2-AR antibody (1:1000 dilution, Abcam, 4 ab182136), phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) antibody (1:1000 dilution, Cell Signaling, #8828), Smad2/3 antibody (1:1000, Cell Signaling, #5678), anti-TβRI antibody (1:200 dilution, Santa Cruz, sc-399), anti-TβRII antibody (1:200 dilution, Santa Cruz,sc-17792), anti-TGF-β antibody (1:1000 dilution, Cell Signaling, #3711), anti-GAPDH antibody (1: 5000 dilution, Abcam, ab8245).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Construct

Journal: Hypertension (Dallas, Tex. : 1979)

Article Title: Type III Transforming Growth Factor-β Receptor Drives Cardiac Hypertrophy Through β-Arrestin2-Dependent Activation of Calmodulin-Dependent Protein Kinase II.

doi: 10.1161/HYPERTENSIONAHA.116.07420

Figure Lengend Snippet: Figure 4. Type III transforming growth factor-β receptor (TβRIII) activates calmodulin-dependent protein kinase II (CaMKII) signaling via enhanced physical interaction with β-arrestin2 in vitro. A and B, Co-immunoprecipitation (Co-IP) data demonstrate that TβRIII and β- arrestin2, β-arrestin2 with CaMKII form physical complexes in neonatal mouse cardiomyocytes (NMCMs); cell lysates and n-IgG served as positive and negative control, respectively (n=3 repeats). C, Representative Co-IP experiments demonstrating the magnitude of TβRIII/β-arrestin2 (a) and β-arrestin2/CaMKII complex (b) in NMCMs under the indicated conditions (n=3 repeats). D, Representative Western blot (a) and summarized (b) data showing relative expression levels of p-CaMKII in NMCMs under the indicated conditions. Isoproterenol (ISO): NMCMs stimulated with ISO; +siTβRIII: NMCMs transfected with TβRIII siRNA + ISO stimulation; +NC (siTβRIII): NMCMs transfected with scrambled TβRIII siRNA to serve as negative control+ISO stimulation; TβRIII: NMCMs transfected with plasmid containing TβRIII cDNA; EV: NMCMs transfected with empty vector pcDNA3.1 to serve as negative control for TβRIII overexpression (n=4; *P<0.05 vs Ctrl, #P<0.05 vs ISO). E, Representative Western blots (a) and summarized data (b) demonstrating knocking down of β- arrestin2, in the TβRIII-overexpressed NMCMs, resulted in a decreased expression of β-myosin heavy chain (β-MHC) and p-CaMKII; +siβ- Arr2: knocking down of β-arrestin2 in TβRIII-overexpressed NMCMs; +NC (β-Arr2): scrambled β-arrestin2 siRNA in TβRIII-overexpressed NMCMs (n=3/group; *P<0.05 vs Ctrl, #P<0.05 vs TβRIII overexpression). F, Cell surface area and (G) protein/DNA ratio measured, respectively, in wild-type NMCMs (Ctrl) and in TβRIII-overexpressed NMCMs either in the absence of KN93 (TβRIII) or in the presence of 1 µmol/L KN93 (+KN93) and vehicle (+dimethyl sulphoxide [DMSO]; n=4/group; *P<0.05 vs Ctrl, #P<0.05 vs TβRIII overexpression). H, Representative Western blots (a) and summarized data (b) demonstrating inhibition of CaMKII resulted in decreased expression of β-MHC and p-CaMKII in the TβRIII-overexpressed NMCMs (n=3/group; *P<0.05 vs Ctrl, #P<0.05 vs TβRIII overexpression). All data presented as mean±SD.

Article Snippet: The primary antibodies used in the present study included anti-TβRIII antibody (1:1000 dilution, Cell signaling #2519; 1:200 dilution, Santa Cruz, sc-28975, sc-6199), anti-β-arrestin2 antibody (1:1000 dilution, Cell Signaling #3857; 1:200 dilution, Santa Cruz, sc-13140), anti-p-CaMKII antibody (1:1000 dilution, Cell Signaling, #3361), anti-CaMKII antibody (1:200 dilution, Santa Cruz, sc-13082), anti-ANP antibody (1: 200 dilution, Santa Cruz, sc-20158), anti-β-MHC (1:5000 dilution, Sigma-Aldrich, M8421), anti-β1-AR antibody (1: 200 dilution, Santa Cruz, sc-568), anti-β2-AR antibody (1:1000 dilution, Abcam, 4 ab182136), phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) antibody (1:1000 dilution, Cell Signaling, #8828), Smad2/3 antibody (1:1000, Cell Signaling, #5678), anti-TβRI antibody (1:200 dilution, Santa Cruz, sc-399), anti-TβRII antibody (1:200 dilution, Santa Cruz,sc-17792), anti-TGF-β antibody (1:1000 dilution, Cell Signaling, #3711), anti-GAPDH antibody (1: 5000 dilution, Abcam, ab8245).

Techniques: In Vitro, Immunoprecipitation, Co-Immunoprecipitation Assay, Negative Control, Western Blot, Expressing, Transfection, Plasmid Preparation, Over Expression, Inhibition

Journal: Hypertension (Dallas, Tex. : 1979)

Article Title: Type III Transforming Growth Factor-β Receptor Drives Cardiac Hypertrophy Through β-Arrestin2-Dependent Activation of Calmodulin-Dependent Protein Kinase II.

doi: 10.1161/HYPERTENSIONAHA.116.07420

Figure Lengend Snippet: Figure 7. Type III transforming growth factor-β receptor (TβRIII) does interact with β1-adrenergic receptor (β1-AR) and β2- AR, and manipulating expression levels of TβRIII does not affect downstream signaling of β1-AR1. Co-immunoprecipitation data demonstrate that TβRIII does not form a complex with either β1-AR (A) or β2-AR (B; n=3 repeats). cAMP (C) and protein kinase A (PKA) (D) levels were not increased in the left ventricular tissues of cardiac- specific transgenic expression of TβRIII mice compared with wild-type (WT) mice (n=5–6). Enhanced level of cAMP (E) and PKA (F) levels in the left ventricle tissues mouse heart by isoproterenol (ISO) infusion were not affected by adeno-associated viral 9 (AAV9)-shRNA–mediated knocking down of TβRIII (n=5). All data presented as mean±SD.

Article Snippet: The primary antibodies used in the present study included anti-TβRIII antibody (1:1000 dilution, Cell signaling #2519; 1:200 dilution, Santa Cruz, sc-28975, sc-6199), anti-β-arrestin2 antibody (1:1000 dilution, Cell Signaling #3857; 1:200 dilution, Santa Cruz, sc-13140), anti-p-CaMKII antibody (1:1000 dilution, Cell Signaling, #3361), anti-CaMKII antibody (1:200 dilution, Santa Cruz, sc-13082), anti-ANP antibody (1: 200 dilution, Santa Cruz, sc-20158), anti-β-MHC (1:5000 dilution, Sigma-Aldrich, M8421), anti-β1-AR antibody (1: 200 dilution, Santa Cruz, sc-568), anti-β2-AR antibody (1:1000 dilution, Abcam, 4 ab182136), phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) antibody (1:1000 dilution, Cell Signaling, #8828), Smad2/3 antibody (1:1000, Cell Signaling, #5678), anti-TβRI antibody (1:200 dilution, Santa Cruz, sc-399), anti-TβRII antibody (1:200 dilution, Santa Cruz,sc-17792), anti-TGF-β antibody (1:1000 dilution, Cell Signaling, #3711), anti-GAPDH antibody (1: 5000 dilution, Abcam, ab8245).

Techniques: Expressing, Immunoprecipitation, Transgenic Assay, shRNA