Journal: Journal of Biological Chemistry

Article Title: Heterodimerization of α2A- and β1-Adrenergic Receptors

doi: 10.1074/jbc.m207968200

Figure Lengend Snippet: FIG. 2. Co-internalization of 1AR with 2AAR. HA-2AAR and FLAG-1AR were expressed either separately (solid bars) or together (striped bars) in HEK-293 cells. The internalization of 2AAR (A) and 1AR (B) was examined using a luminometer-based assay following 10-min stimulations with the -adrenergic agonist isoproterenol (Iso; 10 m), the 2-adrenergic agonist UK 14,304 (UK; 10 M), or a combina- tion of the two agonists together. As shown in A, 2AAR exhibited 15% internalization in response to UK stimulation but no significant inter- nalization in response to isoproterenol under any condition. Conversely, as shown in B, 1AR exhibited 25–30% internalization in response to isoproterenol but also exhibited 15% internalization in response to stimulation with UK. This effect was only observed, however, when 2AAR was coexpressed (** indicates significantly different from 1AR alone, p 0.01). These data suggest that 1AR can co-internalize with agonist-activated 2AAR. The bars and error bars represent the means S.E. for 4–5 independent experiments for each condition, with each experiment being performed in triplicate.

Article Snippet: After another 48 h, cells were treated with varying concentrations of isoproterenol for 10 min and harvested with cell harvest buffer (50 mM Tris, pH 7.4, 250 M Ro 20-1724 (Tocris, Ellisville, NJ), 5 mM MgCl2, 1 mM ATP, and 1 M GTP).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Heterodimerization of α2A- and β1-Adrenergic Receptors

doi: 10.1074/jbc.m207968200

Figure Lengend Snippet: FIG. 3. Immunofluorescence confocal microscopy reveals ago- nist-promoted co-internalization of 2A- and 1-adrenergic re- ceptors. HA-2AAR (red) and FLAG-1AR (green) were co-transfected into HEK-293 cells and visualized using secondary antibodies coupled to rhodamine and FITC, respectively. In the absence of agonist stimu- lation, immunostaining for both receptors was found predominantly in the plasma membrane (A–C). Stimulation with isoproterenol (Iso) for 10 min induced significant mobilization of 1AR inside the cell (D) but had no significant effect on the subcellular distribution of 2AAR (E and F). Stimulation with UK 14,034 (UK), in contrast, resulted in significant internalization of both 2AAR (H) and 1AR (G) and marked co-local- ization of the two receptors in intracellular regions (I, with co-localiza- tion indicated in yellow). The specificity of staining was determined in control (Con) experiments using both untransfected and transfected cells incubated in the absence and presence of the relevant primary antibodies. These data are representative of 3–5 experiments for each condition.

Article Snippet: After another 48 h, cells were treated with varying concentrations of isoproterenol for 10 min and harvested with cell harvest buffer (50 mM Tris, pH 7.4, 250 M Ro 20-1724 (Tocris, Ellisville, NJ), 5 mM MgCl2, 1 mM ATP, and 1 M GTP).

Techniques: Immunofluorescence, Confocal Microscopy, Transfection, Immunostaining, Clinical Proteomics, Membrane, Staining, Control, Incubation

Journal: Journal of Biological Chemistry

Article Title: Heterodimerization of α2A- and β1-Adrenergic Receptors

doi: 10.1074/jbc.m207968200

Figure Lengend Snippet: FIG. 5. Coexpression of 1AR with 2AAR alters the potency of isoproterenol-induced stimulation of adenylyl cyclase. HEK-293 cells were transfected with either 1AR alone (filled squares, solid line) or 1AR/2AAR (open triangles, dotted line). Expression levels of the 1AR were identical for the two transfection conditions, as assessed by Western blot. The cells were stimulated with increasing concentrations of isoproterenol, and agonist-induced rises in cellular cyclic AMP were quantified. The maximal extent of cyclic AMP produced in the 1AR/2AAR cells was 106 8% of that produced in the cells trans- fected with only 1AR. The EC50 for isoproterenol stimulation of 1AR alone was 0.16 0.02 nM, as compared with 0.68 0.17 for 1AR/ 2AAR (significantly different from 1AR alone, p 0.01). The points and error bars represent the means and S.E. values for four independ- ent determinations.

Article Snippet: After another 48 h, cells were treated with varying concentrations of isoproterenol for 10 min and harvested with cell harvest buffer (50 mM Tris, pH 7.4, 250 M Ro 20-1724 (Tocris, Ellisville, NJ), 5 mM MgCl2, 1 mM ATP, and 1 M GTP).

Techniques: Transfection, Expressing, Western Blot, Produced

Journal: PLoS ONE

Article Title: Circulating Fibroblast Growth Factor-2, HIV-Tat, and Vascular Endothelial Cell Growth Factor-A in HIV-Infected Children with Renal Disease Activate Rho-A and Src in Cultured Renal Endothelial Cells

doi: 10.1371/journal.pone.0153837

Figure Lengend Snippet: (A) Phase contrast microscopy picture of cultured HGEc-1, Magnification, X100. (B) Immunohistochemistry staining of cultured HGEc-1 expressing the endothelial cell marker Von Willebrand Factor (vWf), red color, Magnification, X100. Immunofluorescence staining of cultured HGEc-1 for (C) CD31, (D) VEGFR-2, (E) VE-cadherin, all in red color, and (F) human podocytes incubated with the anti- CD31 antibody (negative controls). Cell nuclei are visualized with DAPI in blue color. Scale bar = 20 μm. (G) Western blots show CD31, VEGFR2, VE-cadherin, and Beta actin expression in cultured HGEc-1, human umbilical vein endothelial cells (HUVEC), and human renal embryonic epithelial cells (HEK293). (H) Changes in trans-endothelial electrical resistance (TEER) induced by the cyclic AMP analogue 8-pCPT-cAMP (30 μm) in combination with the cAMP- phosphodiesterase inhibitor RO-20-1724 (20 μm); thrombin (100 units/ml), or VEGF-A (50 ng/ml). Results are expressed as changes relative to controls. Bar graphs show mean ± SEM corresponding to three different experiments. Values significantly different from control were marked with asterisk , *p<0.05 and **p<0.01.

Article Snippet: The reagents described below were obtained from the following sources: human recombinant VEGF-165 (PeproTech (Rocky Hill, NJ); HIV-1 Tat protein (NIH AIDS Reagent Program); human recombinant FGF-2 (R&D Systems); Heparin derived from porcine intestinal mucosa, USP 5,000 USP (Units /ml) APP Pharmaceuticals LLC; SU6656 (Calbiochem); C3 Transferase (List Biological Labs, Campbell, CA); Y-27632, cyclic 3’5 monophosphate (cAMP) analog, thrombin, DAPI and Beta-actin mouse monoclonal antibody (Sigma-Aldrich, MO); RO 20–1724 (MyBiosource San Diego), and RhoA (67B9) rabbit monoclonal antibody, phospo-p44/42 MAP kinase (Thr202/Tyr204), p44/42 MAP kinase, Src rabbit monoclonal antibody, phospho-Myosin Light Chain 2 (Thr/Ser 19) rabbit polyclonal antibody, Myosin Light Chain 2 rabbit polyclonal antibody, and VEGF receptor 2 (VEGFR2) 55B11 rabbit monoclonal antibody, were all obtained from Cell Signaling Technology (Danvers, MA).

Techniques: Microscopy, Cell Culture, Immunohistochemistry, Staining, Expressing, Marker, Immunofluorescence, Incubation, Western Blot

Journal: PLoS Neglected Tropical Diseases

Article Title: The single cyclic nucleotide-specific phosphodiesterase of the intestinal parasite Giardia lamblia represents a potential drug target

doi: 10.1371/journal.pntd.0005891

Figure Lengend Snippet: Potency of selected PDE inhibitors. From those compounds exhibiting IC 50 <100 μM against recombinant GlPDE (aa588-1371) and EC 50 <50 μM against Giardia trophozoites in the benchmark screening (see in Fig 3 ) exact dose responses were determined (values highlighted in green). PDE activity assays were done in triplicates and repeated at least once (SE of logIC 50 < = 0.1), and Giardia cell susceptibility assays were done in quadruplicates and repeated 2 to 5 times (SE of logEC 50 < = 0.15). n.d., not determined.

Article Snippet: PDE inhibitors were from the following sources: isobutyl-methyl-xanthine (IBMX), 8-methoxymethyl-IBMX, vinpocetine, rolipram, dipyridamole, papaverine, BAY 73–6691, BRL 50481 were from Sigma; zardaverine, cilostamide were from BioMol Anawa Trading (Wangen, Switzerland); pentoxifylline was from Calbiochem (Merck Millipore, Schaffhausen, Switzerland); etatolate was from Tocris Bioscience (Lucerna-Chem, Lucerne, Switzerland); erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), milrinone, trequinsin, Ro 20–1724, dipyridamole, zaprinast were obtained from Fisher Scientific; BAY 60–7550 was from Biotrend Chemicals (Destin, FL, USA), sildenafil citrate was generously provided by Pfizer, Inc.

Techniques: Recombinant, Activity Assay