rabbit polyclonal anti gipr  (Cusabio)

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: GIP Receptor Antagonism Eliminates Paradoxical Growth Hormone Secretion in Some Patients With Acromegaly

doi: 10.1210/clinem/dgae583

Figure Lengend Snippet: Representative images of adenomas with normal and abundant GIPR expression. Normal GIPR (glucose-dependent insulinotropic polypeptide receptor) expression visualized by immunohistochemistry in a nonparadoxical patient by A) brightfield microscopy, and C) and D) fluorescence microscopy. Abundant GIPR expression visualized in a responder by E, brightfield microscopy, and G and H, fluorescence microscopy. B) and F) Growth hormone (GH) visualization in red; C) and G) GIPR visualization in green; and D) and H) merged red and green staining showing GIPR colocalization with GH in the somatotrophs. All images were acquired with 40× magnification. Scale bars (white), 20 μm.

Article Snippet: Sections were preincubated for 10 minutes in 2% bovine serum albumin and incubated at 4 °C overnight with a rabbit anti-GIPR polyclonal antibody (1:100; catalog No. PA5-33585, RRID:AB_2550965, Thermo Fisher Scientific) and a mouse anti-GH monoclonal antibody (1:1000; catalog No. SC-374266, RRID:AB_10989917, Santa Cruz Biotechnology).

Techniques: Expressing, Immunohistochemistry, Microscopy, Fluorescence, Staining

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: GIP Receptor Antagonism Eliminates Paradoxical Growth Hormone Secretion in Some Patients With Acromegaly

doi: 10.1210/clinem/dgae583

Figure Lengend Snippet: Effect of GIP on GH secretion ex vivo and in vitro and on cAMP accumulation in vitro. A) GH secretion from somatotropinoma-derived primary cultures expressed as relative secretion from vehicle. Light blue bars, GIP 10 nM stimulation; dark blue bars, GIP 100 nM stimulation; black arrows, confirmed paradoxical GH secretion to OGTT. B) GIP dose response of cAMP accumulation in hGIPR transiently transfected GH3 cells. Results are expressed as relative cAMP accumulation compared to the maximal GIP-induced cAMP response. Solid line, closed circles: hGIPR vector; dashed line, open squares: empty pCMV vector. C) GIP and GIP + GIPR antagonist-induced GH secretion from hGIPR transiently transfected GH3 cells expressed as relative secretion with vehicle. Data are presented as mean ± SEM. One-way analysis of variance with Holm-Šídák multiple comparison test was used to assess statistical significance in the GH3 cell line, and an unpaired t test to assess statistical significance in primary cultures. * P less than .05 increase from vehicle. Ψ P less than .05 decrease from GIP-stimulated cells.

Article Snippet: Sections were preincubated for 10 minutes in 2% bovine serum albumin and incubated at 4 °C overnight with a rabbit anti-GIPR polyclonal antibody (1:100; catalog No. PA5-33585, RRID:AB_2550965, Thermo Fisher Scientific) and a mouse anti-GH monoclonal antibody (1:1000; catalog No. SC-374266, RRID:AB_10989917, Santa Cruz Biotechnology).

Techniques: Ex Vivo, In Vitro, Derivative Assay, Transfection, Plasmid Preparation, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Molecular basis of signal transduction mediated by the human GIPR splice variants

doi: 10.1073/pnas.2306145120

Figure Lengend Snippet: Cryo-EM structures of SV1 and SV2 of GIPR in complex with G s . ( A ) Cryo-EM map ( Left ) and structural model ( Right ) of SV1 in complex with G s . ( B ) Cryo-EM map ( Left ) and structural model ( Right ) of SV2 in complex with G s . SV1 is shown in hot pink, SV2 in cornflower blue, Gα s in medium slate blue, Gβ subunit in light salmon, Gγ subunit in olive, and Nb35 in medium purple.

Article Snippet: The PVDF membrane was blocked by protein-free rapid blocking buffer (EpiZyme Biotec) and incubated with rabbit polyclonal antibody against GIPR (Abcam) and rabbit antibody against Na, K-ATPase (Cell Signaling Technology) followed by three washes with Tris-buffered saline with 0.1% Tween 20 (TBST) (EpiZyme).

Techniques: Cryo-EM Sample Prep

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Molecular basis of signal transduction mediated by the human GIPR splice variants

doi: 10.1073/pnas.2306145120

Figure Lengend Snippet: Ligand-binding, signaling profiles and cell surface expression of GIPR and its SVs. ( A ) Competitive inhibition of 125 I-GIP 1-42 binding to GIPR and SVs by unlabeled GIP 1-42 . Binding affinity is quantified by reduction of radioactivity (counts per minute, CPM). ( B ) Concentration–response curves of cAMP accumulation elicited by GIP 1-42 at GIPR and SVs. ( C and D ) β-arrestins 1 (β-arr1) and 2 (β-arr2) recruitment by GIPR and SVs. Concentration–response characteristics are shown as the area-under-the-curve (AUC) across the time course–response curve (0 to 10 min) for each concentration. Data shown are means ± SEM of at least three independent experiments (n = 3 to 5) performed in quadruplicate (cAMP accumulation) or duplicate (specific binding and β-arrestin recruitment). Signals were normalized to the maximum (max) response of the WT GIPR, and concentration–response curves were analyzed using a three-parameter logistic equation. ( E ) Cell surface expression of GIPR and each SV by western blot using antibodies against GIPR and Na, K-ATPase (internal reference), Left panel. The Right panel shows the quantification. Data shown are means ± SEM of three independent experiments (n = 3). One-way ANOVA was used to determine statistical difference (* P < 0.05).

Article Snippet: The PVDF membrane was blocked by protein-free rapid blocking buffer (EpiZyme Biotec) and incubated with rabbit polyclonal antibody against GIPR (Abcam) and rabbit antibody against Na, K-ATPase (Cell Signaling Technology) followed by three washes with Tris-buffered saline with 0.1% Tween 20 (TBST) (EpiZyme).

Techniques: Ligand Binding Assay, Expressing, Inhibition, Binding Assay, Radioactivity, Concentration Assay, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Molecular basis of signal transduction mediated by the human GIPR splice variants

doi: 10.1073/pnas.2306145120

Figure Lengend Snippet: Structural comparison of GIPR, SV1, and SV2. ( A ) Structural comparison of GIP–GIPR–G s (PDB code: 7DTY), SV1–G s , and SV2–G s . The structures of SV1 and SV2 are superimposed on the GIP-bound GIPR using the Cα carbons of the TMD residues. ( B ) Extracellular view ( Left ) and intracellular view ( Right ) of the receptor TMD. The ECD of GIPR and all the G proteins are omitted for clarity. The receptors and peptides are colored as labeled. ( C ) Surface representation of the TMD peptide-binding pocket among GIPR, SV1, and SV2 structures. The GIP is shown in orange, the N-terminal half of TMD (TMs 1-5, ECL1, and ECL2) in dodger blue, and the C-terminal half of TMD (TM5, ECL3, and TM6) in cyan.

Article Snippet: The PVDF membrane was blocked by protein-free rapid blocking buffer (EpiZyme Biotec) and incubated with rabbit polyclonal antibody against GIPR (Abcam) and rabbit antibody against Na, K-ATPase (Cell Signaling Technology) followed by three washes with Tris-buffered saline with 0.1% Tween 20 (TBST) (EpiZyme).

Techniques: Comparison, Labeling, Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Molecular basis of signal transduction mediated by the human GIPR splice variants

doi: 10.1073/pnas.2306145120

Figure Lengend Snippet: Molecular interactions within the TM6/ECL3/TM7-TM1/TM3/ECL2/TM5 interfaces of GIPR, SV1, and SV2. ( A ) The extracellular halves of TM6 of both SV1 and SV2 fold inward to form massive polar and nonpolar interactions with TM3, ECL2, and TM5. The structures of SV1 and SV2 are superimposed on the GIP-bound GIPR (PDB code: 7DTY) using the Cα carbons of the TMD residues. ( B ) The ECL3 and extracellular half of TM7 of SV1 and SV2 are clasped by TM1, TM3 and ECL2 with the formation of multiple contacts.

Article Snippet: The PVDF membrane was blocked by protein-free rapid blocking buffer (EpiZyme Biotec) and incubated with rabbit polyclonal antibody against GIPR (Abcam) and rabbit antibody against Na, K-ATPase (Cell Signaling Technology) followed by three washes with Tris-buffered saline with 0.1% Tween 20 (TBST) (EpiZyme).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Molecular basis of signal transduction mediated by the human GIPR splice variants

doi: 10.1073/pnas.2306145120

Figure Lengend Snippet: Effects of SVs on ligand binding and GIPR-mediated signal transduction in HEK293T cells coexpressing GIPR and individual SVs

Article Snippet: The PVDF membrane was blocked by protein-free rapid blocking buffer (EpiZyme Biotec) and incubated with rabbit polyclonal antibody against GIPR (Abcam) and rabbit antibody against Na, K-ATPase (Cell Signaling Technology) followed by three washes with Tris-buffered saline with 0.1% Tween 20 (TBST) (EpiZyme).

Techniques: Ligand Binding Assay, Transduction