d2r expression  (R&D Systems)

Journal: Endocrine Pathology

Article Title: Clinical and Molecular Characteristics of Gonadotroph Pituitary Tumors According to the WHO Classification

doi: 10.1007/s12022-023-09794-w

Figure Lengend Snippet: Gene expression data obtained in 54 GnPT: correlation matrix

Article Snippet: Two commercial antibodies were tested for D2R expression: a monoclonal antibody (mab9266–100, R&D system, distributed by Aurogene, Italy, referred to as D2R-mAb, dilution 1:100) and a polyclonal antibody (AB5084P, Sigma-Aldrich, distributed by Sial, Italy, referred to as D2R-pAb, dilution 1:500), the latter being previously used in this indication [ , ].

Techniques: Gene Expression

Journal: Endocrine Pathology

Article Title: Clinical and Molecular Characteristics of Gonadotroph Pituitary Tumors According to the WHO Classification

doi: 10.1007/s12022-023-09794-w

Figure Lengend Snippet: Bio-clinical and molecular characteristics of GnPT according to the presence or the absence of gonadotropin immunostaining

Article Snippet: Two commercial antibodies were tested for D2R expression: a monoclonal antibody (mab9266–100, R&D system, distributed by Aurogene, Italy, referred to as D2R-mAb, dilution 1:100) and a polyclonal antibody (AB5084P, Sigma-Aldrich, distributed by Sial, Italy, referred to as D2R-pAb, dilution 1:500), the latter being previously used in this indication [ , ].

Techniques: Gene Expression, Immunohistochemistry

Journal: Endocrine Pathology

Article Title: Clinical and Molecular Characteristics of Gonadotroph Pituitary Tumors According to the WHO Classification

doi: 10.1007/s12022-023-09794-w

Figure Lengend Snippet: D2R gene expression in GnPT and functional lactotroph tumors. No significant difference in D2R expression was found between functional lactotroph tumors (PRL) and GnPT, or between FSH/LH and pSF1 phenotypes ( A ). In contrast, D2R transcripts were significantly lower in recurrent GnPT as compared to non-recurrent cases ( B )

Article Snippet: Two commercial antibodies were tested for D2R expression: a monoclonal antibody (mab9266–100, R&D system, distributed by Aurogene, Italy, referred to as D2R-mAb, dilution 1:100) and a polyclonal antibody (AB5084P, Sigma-Aldrich, distributed by Sial, Italy, referred to as D2R-pAb, dilution 1:500), the latter being previously used in this indication [ , ].

Techniques: Gene Expression, Functional Assay, Expressing

Journal: Endocrine Pathology

Article Title: Clinical and Molecular Characteristics of Gonadotroph Pituitary Tumors According to the WHO Classification

doi: 10.1007/s12022-023-09794-w

Figure Lengend Snippet: D2R immunostaining in controls and representative examples of GnPT using a monoclonal (D2R-mAb) or a polyclonal (D2R-pAb) antibody. In the brain tissue ( A , B ) (×200 magnification; inset at ×400), D2R staining was essentially cytoplasmic. In normal pituitary gland fragments, nuclear ( C ) or cytoplasmic with occasional membrane ( D ) D2R staining was observed (×200 magnification; inset at ×400). Similar results were obtained on functional lactotroph tumors ( E , F ) (×200 magnification; inset at ×400). Representative examples of D2R immunostaining in GnPT are also shown ( G , H ) using the mAb ( G ) nuclear staining which was observed in a normal juxta-tumoral pituitary fragment (indicated by a star) and in scattered neoplastic cells (inset) (×100 magnification; inset at ×400), whereas cytoplasmic staining was focally observed with the pAb (H) (×400 magnification)

Article Snippet: Two commercial antibodies were tested for D2R expression: a monoclonal antibody (mab9266–100, R&D system, distributed by Aurogene, Italy, referred to as D2R-mAb, dilution 1:100) and a polyclonal antibody (AB5084P, Sigma-Aldrich, distributed by Sial, Italy, referred to as D2R-pAb, dilution 1:500), the latter being previously used in this indication [ , ].

Techniques: Immunostaining, Staining, Membrane, Functional Assay

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: Investigation of D2R internalization in non-neuronal cell systems. (A) HEK293 cells were transiently transfected with D2R-Rluc8 and a modified GFP10, which together produce constitutive bystander BRET. On the day of assay, the cells were stimulated with either dopamine (DA) or MLS1547 for 30 min at 37°C before BRET measurement. The data are graphed as mean dose-response curves from three independent experiments. DA exhibited an logEC50 of −7.00 ± 0.36 (EC50, 0.1 μM) for BRET reduction/D2R internalization, while MLS1547 treatment failed to alter the BRET signal. (B) Maximum BRET signal obtained after treatment with vehicle (control, 0.81 ± 0.02), 10 μM DA (0.65 ± 0.02), or 30 μM MLS1547 (0.79 ± 0.03). All data are expressed as mean ± SEM of the BRET ratio from three independent experiments. * P < 0.05, unpaired Student's t -test compared to untreated controls. (C) HA-tagged D2R was transiently transfected into HEK293 cells and surface D2R was biotinylated as described in Methods. After washing, cells were treated with either vehicle (control), 10 μM quinpirole, or 10 μM MLS1547 or for 30 min at 37°C. Biotin was cleaved from D2R that remained on the cell surface, followed by cell lysis and isolation of biotinylated (internalized) D2R using avidin beads. Western blotting was used to detect D2R and also transferrin receptor (TfR) (as a gel loading control). A single experiment, representative of three, is shown (the original full blot is displayed in the ). (D) Densitometric analyses were performed and the data expressed as D2R/TfR ratios, relative to the untreated controls (1.0). Treatment values are provided as mean ± S.E.M: quinpirole = 2.8 ± 0.2 and MLS1547 = 1.5 ± 0.03. One-way ANOVA followed by Bonferroni's post-test was performed: * p < 0.05, and ** p < 0.01.

Article Snippet: Briefly, CHO-K1 cells expressing the D2R long isoform (DiscoverX, Inc.) were maintained in Ham's F12 media supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 20 mM HEPES, and 800 μg/ml G418 and 300 μg/ml hygromycin at 37°C, 5% CO 2 , and 90% humidity.

Techniques: Transfection, Modification, Control, Lysis, Isolation, Avidin-Biotin Assay, Western Blot

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: Investigation of D2R internalization in neuronal cell systems. (A) Striatal neurons were prepared and transfected with the D2R-pHluorin construct (pH-DRD2) as described in Methods. 48 h later, the neurons were treated with vehicle (control) or 10 μM dopamine (DA), 30 μM MLS1547, or 30 μM sulpiride for 45 min. Neurons were washed and then stained with a polyclonal GFP antibody to detect surface D2R followed by fixation, permeabilization and re-staining with a monoclonal GFP antibody to detect total cellular D2R. The ratios between surface to total pH-DRD2 signal were normalized to the control group in each experiment and are expressed as mean ± S.E.M of the indicated number of cells ( n ): control = 1.00 ± 0.02, n = 746; DA = 0.72 ± 0.02, n = 627; MLS1547 = 0.90 ± 0.03, n = 812, sulpiride = 1.05 ± 0.08, n = 171. One-way ANOVA followed by Bonferroni's post-test was performed: * p < 0.0001 and ** p < 0.0262. (B) Striatal neurons expressing the D2R-pHluorin construct were exposed to vehicle (control) or 10 μM DA, 30 μM MLS1547, or 30 μM sulpiride for 20 min and the number of reinsertion events/(min × μm 2 ) were visualized in real time as described in Methods. The average reinsertion frequencies for the drug treatments were normalized to the control group in each experiment and are expressed as mean ± S.E.M of the indicated number of cells ( n ): control = 1.00 ± 0.20, n = 51; DA = 5.70 ± 0.54, n = 49; MLS1547 = 1.73 ± 0.22, n = 50, sulpiride = 1.56 ± 0.34, n = 15. One-way ANOVA followed by Bonferroni's post-test was performed, * p < 0.0001.

Article Snippet: Briefly, CHO-K1 cells expressing the D2R long isoform (DiscoverX, Inc.) were maintained in Ham's F12 media supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 20 mM HEPES, and 800 μg/ml G418 and 300 μg/ml hygromycin at 37°C, 5% CO 2 , and 90% humidity.

Techniques: Transfection, Construct, Control, Staining, Expressing

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: A halogen moiety in area 1 on ring B of MLS1547 enables G protein-bias. CHO cells stably expressing the D2R were assayed for agonist-mediated inhibition of forskolin-stimulated cAMP accumulation (A) or recruitment of β-arrestin to the D2R (B) , as described in the Materials and Methods. Representative experiments are shown with averaged curve parameters derived from at least three separate experiments displayed in Table . Data are expressed as a percentage of the maximum response to dopamine, which was assessed in each experiment.

Article Snippet: Briefly, CHO-K1 cells expressing the D2R long isoform (DiscoverX, Inc.) were maintained in Ham's F12 media supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 20 mM HEPES, and 800 μg/ml G418 and 300 μg/ml hygromycin at 37°C, 5% CO 2 , and 90% humidity.

Techniques: Stable Transfection, Expressing, Inhibition, Derivative Assay

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: A hydroxyl group in area 2 on ring B of MLS1547 is required for both potency and efficacy of G protein signaling. CHO cells stably expressing the D2R were assayed for agonist-mediated inhibition of forskolin-stimulated cAMP accumulation (A) or recruitment of β-arrestin to the D2R (B) , as described in the Materials and Methods. Representative experiments are shown with averaged curve parameters derived from at least three separate experiments displayed in Table . Data are expressed as a percentage of the maximum response to dopamine, which was assessed in each experiment.

Article Snippet: Briefly, CHO-K1 cells expressing the D2R long isoform (DiscoverX, Inc.) were maintained in Ham's F12 media supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 20 mM HEPES, and 800 μg/ml G418 and 300 μg/ml hygromycin at 37°C, 5% CO 2 , and 90% humidity.

Techniques: Stable Transfection, Expressing, Inhibition, Derivative Assay

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: Effects of combined modifications of areas 1 and 2 of ring B of MLS1547 on signaling bias. CHO cells stably expressing the D2R were assayed for agonist-mediated inhibition of forskolin-stimulated cAMP accumulation (A) or recruitment of β-arrestin to the D2R (B) , as described in the Materials and Methods. Representative experiments are shown with averaged curve parameters derived from at least three separate experiments displayed in Table . Data are expressed as a percentage of the maximum response to dopamine, which was assessed in each experiment.

Article Snippet: Briefly, CHO-K1 cells expressing the D2R long isoform (DiscoverX, Inc.) were maintained in Ham's F12 media supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 20 mM HEPES, and 800 μg/ml G418 and 300 μg/ml hygromycin at 37°C, 5% CO 2 , and 90% humidity.

Techniques: Stable Transfection, Expressing, Inhibition, Derivative Assay

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: A hydrophobic moiety in the correct orientation is integral to G protein biased signaling activity of the MLS1547 scaffold. CHO cells stably expressing the D2R were assayed for agonist-mediated inhibition of forskolin-stimulated cAMP accumulation (A) or recruitment of β-arrestin to the D2R (B) , as described in the Materials and Methods. Representative experiments are shown with averaged curve parameters derived from at least three separate experiments displayed in Table . Data are expressed as a percentage of the maximum response to dopamine, which was assessed in each experiment.

Article Snippet: Briefly, CHO-K1 cells expressing the D2R long isoform (DiscoverX, Inc.) were maintained in Ham's F12 media supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 20 mM HEPES, and 800 μg/ml G418 and 300 μg/ml hygromycin at 37°C, 5% CO 2 , and 90% humidity.

Techniques: Activity Assay, Stable Transfection, Expressing, Inhibition, Derivative Assay

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: The addition of a second nitrogen at the 6-position of ring D increases agonist potency, but decreases G protein signaling bias. CHO cells stably expressing the D2R were assayed for agonist-mediated inhibition of forskolin-stimulated cAMP accumulation (A) or recruitment of β-arrestin to the D2R (B) , as described in the Materials and Methods. Representative experiments are shown with averaged curve parameters derived from at least three separate experiments displayed in Table . Data are expressed as a percentage of the maximum response to dopamine, which was assessed in each experiment.

Article Snippet: Briefly, CHO-K1 cells expressing the D2R long isoform (DiscoverX, Inc.) were maintained in Ham's F12 media supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 20 mM HEPES, and 800 μg/ml G418 and 300 μg/ml hygromycin at 37°C, 5% CO 2 , and 90% humidity.

Techniques: Stable Transfection, Expressing, Inhibition, Derivative Assay