somatostatin r2/sstr2 antibody  (Bio-Techne corporation)

Journal: Molecular Endocrinology

Article Title: Ligand-Dependent Mechanisms of sst2A Receptor Trafficking: Role of Site-Specific Phosphorylation and Receptor Activation in the Actions of Biased Somatostatin Agonists

doi: 10.1210/me.2010-0398

Figure Lengend Snippet: Agonist-specific effects on sst2A receptor internalization in pancreatic cells. AR42J cells were incubated at 37 C with 100 nm SS14, 1 μm SOM230, or 1 μm KE108 for the times shown. After agonist treatment, cells were rinsed with cold PBS, fixed, and permeabilized as described in Materials and Methods. sst2A receptors were immuno-labeled with receptor antibody followed with DTAF-conjugated donkey antirabbit IgG, and receptor distribution was then assessed by confocal microscopy. The images shown are representative of results from at least three independent experiments. The right panel of each pair of images shows an enlargement of a typical cell observed in the left panel. Although SS14 elicits pronounced receptor internalization within 2 min, SOM230 and KE108 have a much smaller effect on receptor endocytosis with substantial membrane staining still evident after 15 min of peptide treatment.

Article Snippet: Phosphorylation-independent sst2A receptor antibodies (NB100-74537) were purchased from Novus Biologicals (Littleton, CO).

Techniques: Incubation, Labeling, Confocal Microscopy, Membrane, Staining

Journal: Molecular Endocrinology

Article Title: Ligand-Dependent Mechanisms of sst2A Receptor Trafficking: Role of Site-Specific Phosphorylation and Receptor Activation in the Actions of Biased Somatostatin Agonists

doi: 10.1210/me.2010-0398

Figure Lengend Snippet: Agonist-specific effects on sst2A receptor signaling and internalization in CHO cells. A, CHO-sst2A cells, preincubated with [3H]adenine for 3 h at 37 C, were subsequently incubated with 1 mm IBMX for 5 min. Cells were then treated for 10 min at 37 C with 10 μm forskolin in the presence of different concentrations of SS14 (●), KE108 (▴), or SOM230 (■). Reactions were stopped by the addition of 5% trichloroacetic acid, and intracellular cAMP was measured as described in Materials and Methods. Cyclic AMP in each group is expressed as a percentage of that measured in cells treated with forskolin alone. B and C, CHO-sst2A cells were preincubated with HA antibody to label cell surface receptors and then treated at 37 C with varying concentrations of SS14 (●), KE108 (▴), or SOM230 (■) for 30 min (B) or with 100 nm SS14 (●), 1 μm KE108 (▴), or 1 μm SOM230 (■) for varying times (C). After fixation with paraformaldehyde, surface receptors were quantitated by ELISA as described in Materials and Methods. Receptors remaining at the cell surface after peptide treatment are expressed as a percentage of untreated control cells. Dose-response data were fit to a one-component sigmoidal curve using least squares nonlinear regression analysis with GraphPad Prism. The rates of receptor internalization were calculated using nonlinear regression curve fitting to a one-phase exponential decay. Each panel shows the mean ± sem from multiple wells in a single experiment and is representative of at least three independent experiments summarized in Tables 1 and ​and2.2. Where not shown, error bars fell within the size of the symbols.

Article Snippet: Phosphorylation-independent sst2A receptor antibodies (NB100-74537) were purchased from Novus Biologicals (Littleton, CO).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: Molecular Endocrinology

Article Title: Ligand-Dependent Mechanisms of sst2A Receptor Trafficking: Role of Site-Specific Phosphorylation and Receptor Activation in the Actions of Biased Somatostatin Agonists

doi: 10.1210/me.2010-0398

Figure Lengend Snippet: Dose dependence for somatostatin analog-induced sst2A receptor signaling and internalization

Article Snippet: Phosphorylation-independent sst2A receptor antibodies (NB100-74537) were purchased from Novus Biologicals (Littleton, CO).

Techniques: Inhibition

Journal: Molecular Endocrinology

Article Title: Ligand-Dependent Mechanisms of sst2A Receptor Trafficking: Role of Site-Specific Phosphorylation and Receptor Activation in the Actions of Biased Somatostatin Agonists

doi: 10.1210/me.2010-0398

Figure Lengend Snippet: Analog-induced sst2A receptor internalization

Article Snippet: Phosphorylation-independent sst2A receptor antibodies (NB100-74537) were purchased from Novus Biologicals (Littleton, CO).

Techniques:

Journal: Molecular Endocrinology

Article Title: Ligand-Dependent Mechanisms of sst2A Receptor Trafficking: Role of Site-Specific Phosphorylation and Receptor Activation in the Actions of Biased Somatostatin Agonists

doi: 10.1210/me.2010-0398

Figure Lengend Snippet: Agonists-specific patterns of sst2A receptor phosphorylation. A, CHO-sst2A cells were incubated at 37 C for 30 min with 10 nm SS14, KE108, or SOM230. Cells were then solubilized, and equal protein samples from each treatment group were purified by adsorption to wheat germ agglutinin-agarose followed by SDS-PAGE and sequential immunoblotting with the phosphosite-specific antibodies shown and then with HA antibody to show receptor loading. B and C, CHO-sst2A cells were incubated at 37 C for 30 min with varying concentration of SS14 (●), KE108 (▴), or SOM230 (■). Cells were then fixed, and receptor phosphorylation at Ser341/343 (B) or Thr353/354 (C) was measured by whole-cell ELISA using phosphosite-specific antibodies. Receptor phosphorylation is expressed as a percent of the response produced by 1 μm SS14. Nonlinear regression curve fitting was performed using GraphPad Prism. Each panel shows the mean ± sem from a single experiment and is representative of at least three independent experiments, summarized in Table 3.

Article Snippet: Phosphorylation-independent sst2A receptor antibodies (NB100-74537) were purchased from Novus Biologicals (Littleton, CO).

Techniques: Phospho-proteomics, Incubation, Purification, Adsorption, SDS Page, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Produced

Journal: Molecular Endocrinology

Article Title: Ligand-Dependent Mechanisms of sst2A Receptor Trafficking: Role of Site-Specific Phosphorylation and Receptor Activation in the Actions of Biased Somatostatin Agonists

doi: 10.1210/me.2010-0398

Figure Lengend Snippet: Site-specific stimulation of sst2A receptor phosphorylation by somatostatin analogs

Article Snippet: Phosphorylation-independent sst2A receptor antibodies (NB100-74537) were purchased from Novus Biologicals (Littleton, CO).

Techniques: Phospho-proteomics

Journal: Molecular Endocrinology

Article Title: Ligand-Dependent Mechanisms of sst2A Receptor Trafficking: Role of Site-Specific Phosphorylation and Receptor Activation in the Actions of Biased Somatostatin Agonists

doi: 10.1210/me.2010-0398

Figure Lengend Snippet: Agonist selective trafficking of the sst2A receptor and EGFP-β-arrestin-2. CHO-K1 cells were transfected with HA-tagged sst2A receptor and EGFP-β-arrestin-2 as described in Materials and Methods and grown for 48 h. Cells were then incubated with HA antibody for 2 h on ice to label cell surface receptors, washed to remove unbound antibody, and treated with 100 nm SS14, 1 μm SOM230, or 1 μm KE108 at 37 C for the times shown. After fixation and permeabilization, cells were incubated with Alexa Fluor 568-conjugated antimouse IgG. Images of antibody-labeled sst2A receptors (red) and EGFP-tagged arrestin (green) from the same cell were acquired with a Nikon A1 confocal microscope. The merged images depict receptor and β-arrestin-2 colocalization (yellow). Shown are representative cells from at least three independent experiments.

Article Snippet: Phosphorylation-independent sst2A receptor antibodies (NB100-74537) were purchased from Novus Biologicals (Littleton, CO).

Techniques: Transfection, Incubation, Labeling, Microscopy

Journal: Molecular Endocrinology

Article Title: Ligand-Dependent Mechanisms of sst2A Receptor Trafficking: Role of Site-Specific Phosphorylation and Receptor Activation in the Actions of Biased Somatostatin Agonists

doi: 10.1210/me.2010-0398

Figure Lengend Snippet: Agonist-induced membrane translocation of β-arrestin-2. CHO cells were transiently transfected with FLAG-β-arrestin-2 and sst2A receptor. After 48 h, cells were incubated at 37 C with 100 nm SS14, 1 μm SOM230, or 1 μm KE108 for the times shown. After cell lysis, membranes were purified by centrifugation as described in Materials and Methods, and the membrane fraction was subjected to SDS-PAGE. Membrane-associated β-arrestin-2 and actin were detected by incubating each blot with anti-FLAG and antiactin antibodies. Band intensities were quantitated from scanned autoradiograms using Scion Image (version 1.63). A, Time course of arrestin translocation to membranes after peptide treatment in representative experiments. The actin band provides a loading control. B, Direct comparison of membrane-associated β-arrestin-2 after treatment of cells with 100 nm SS14, 1 μm SOM230, or 1 μm KE108 for 5 min in a representative experiment. C, Mean ± sem (n = 6) for membrane-bound arrestin after treatment of cells for 5 min with different analogs in experiments like the one shown in B. The ratio of arrestin to actin was calculated in each sample and then expressed as a percentage of the arrestin/actin ratio observed in samples treated with SS14 for 5 min.

Article Snippet: Phosphorylation-independent sst2A receptor antibodies (NB100-74537) were purchased from Novus Biologicals (Littleton, CO).

Techniques: Membrane, Translocation Assay, Transfection, Incubation, Lysis, Purification, Centrifugation, SDS Page, Control, Comparison

Journal: Molecular Endocrinology

Article Title: Ligand-Dependent Mechanisms of sst2A Receptor Trafficking: Role of Site-Specific Phosphorylation and Receptor Activation in the Actions of Biased Somatostatin Agonists

doi: 10.1210/me.2010-0398

Figure Lengend Snippet: Rate of sst2A receptor recycling and dephosphorylation after treatment with different agonists. CHO-sst2A cells were incubated for 30 min at 37 C in the absence of agonist or in the presence of 100 nm SS14 (●), 1 μm KE108 (▴), or 1 μm SOM230 (■). After peptide treatment, cells were washed and incubated at 37 C in fresh media without agonist but in the presence of 100 nm of the antagonist Coy-14. A, After different periods of recovery, cells were fixed with paraformaldehyde, and cell surface receptors were quantitated with anti-HA antibodies by ELISA as described in Materials and Methods. Receptors in treated samples were expressed as a percentage of receptors in untreated control cultures. Shown are replicate samples from a single experiment. Where not shown, error bars fell within the size of the symbols. B and C, After the incubation with antagonist, cells were fixed, and receptor phosphorylation was measured at Ser341/343 (B) or Thr353/354 (C) using the phosphosite-specific antibodies in a whole-cell ELISA. Receptor phosphorylation in treated samples was expressed as a percentage of the phosphorylation observed after treatment with SS14 for 30 min. The graphs represent the mean ± sem of triplicate samples in a representative experiment. In all three panels, rates of receptor dephosphorylation or receptor recovery were calculated using nonlinear regression curve fitting to a one-phase exponential curve with GraphPad Prism. Results from three to six independent experiments are summarized in Table 4.

Article Snippet: Phosphorylation-independent sst2A receptor antibodies (NB100-74537) were purchased from Novus Biologicals (Littleton, CO).

Techniques: De-Phosphorylation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Control, Phospho-proteomics

Journal: Molecular Endocrinology

Article Title: Ligand-Dependent Mechanisms of sst2A Receptor Trafficking: Role of Site-Specific Phosphorylation and Receptor Activation in the Actions of Biased Somatostatin Agonists

doi: 10.1210/me.2010-0398

Figure Lengend Snippet: Rates of analog-induced sst2A receptor recycling and dephosphorylation

Article Snippet: Phosphorylation-independent sst2A receptor antibodies (NB100-74537) were purchased from Novus Biologicals (Littleton, CO).

Techniques: De-Phosphorylation Assay

Journal: Molecular Endocrinology

Article Title: Ligand-Dependent Mechanisms of sst2A Receptor Trafficking: Role of Site-Specific Phosphorylation and Receptor Activation in the Actions of Biased Somatostatin Agonists

doi: 10.1210/me.2010-0398

Figure Lengend Snippet: Rate of agonist-induced internalization of a phosphorylation negative sst2A receptor mutant. CHO cells stably expressing the phosphorylation negative sst2A receptor, Phos−/sst2A, were incubated for the times shown at 37 C with 100 nm SS14 (●), 1 μm KE108 (▴), or 1 μm SOM230 (■). Cell surface receptors were then measured by ELISA as described in Materials and Methods and expressed as a percent of untreated control cells. Shown is the mean ± sem of cell surface receptors determined in replicate samples in a single experiment, representative of two to five independent experiments. Where not visible, error bars fell within the symbols. Nonlinear regression curve fitting to a one-phase exponential decay was performed using GraphPad Prism. The half-times for receptor internalization from multiple independent experiments was 32.9 ± 3.6 min with SS14 (n = 5) and greater than 10 h with KE108 and SOM230.

Article Snippet: Phosphorylation-independent sst2A receptor antibodies (NB100-74537) were purchased from Novus Biologicals (Littleton, CO).

Techniques: Phospho-proteomics, Mutagenesis, Stable Transfection, Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Control