1. Neurotensin receptor–1 and –3 complex modulates the cellular signaling of neurotensin in the HT29 cell line 
Stéphane Martin, Valérie Navarro, Jean Pierre Vincent, Jean Mazella 
Gastroenterology 
Volume 123, Issue 4, Pages (October 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Identification and coimmunoprecipitation of NT receptors endogenously expressed in HT29 cells. (A) HT29 cells were incubated with 0.5 nmol/L azido-125INT for 40 minutes at 37°C in the presence or absence of 1 μmol/L unlabeled NT before ultraviolet irradiation. Homogenates prepared from these cells were analyzed by SDS-PAGE and the radiolabeled bands were detected by using a phosphorimager. (B and C) Solubilized proteins (30 μg) prepared from HT29 cells were transferred onto nitrocellulose after SDS-PAGE and immunoblotted using (B) a rabbit anti-NTR3 antibody or (C) a goat anti-NTR1 antibody (C). (D) HT29 cells pretreated or not with 1 μg × mL−1 tunicamycin were photolabeled with 0.5 nmol/L azido-125INT as described in A. Finally, cell-solubilized extracts were immunoprecipitated by using the anti-NTR3 antibody, analyzed by SDS-PAGE, and autoradiographed.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

3. Fig. 2
NT-induced distribution of cell surface and intracellular heterodimers in HT29 cells. HT29 cells were incubated with 0.5 nmol/L azido-125INT for 10–40 minutes at 37°C followed or not by an acidic wash to remove the ligand bound to the cell surface and then ultraviolet irradiated before solubilization and immunoprecipitation with the anti-NTR3 antibody. (A) Autoradiography of pelleted proteins is shown. (B and C) HT29 cells were incubated at 37°C with 10 nmol/L NT for the indicated times, then cell surface proteins were biotinylated before solubilization as described in the Materials and Methods section. After immunoprecipitation using the anti-NTR3 antibody, proteins were immunoblotted by using (B) HRP-streptavidin, or (C) the anti-NTR1 antibody, or (D) the anti-NTR3 antibody.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Confocal microscopy visualization of cell-surface heterodimers. HT29 cells were incubated with the goat anti-NTR1 (B) and the rabbit anti-NTR3 (A) antibodies. Cells were then fixed and stained with both a fluorescein isothiocyanate–conjugated donkey anti-goat and a Texas red-conjugated donkey anti-rabbit antibodies, and analyzed by confocal microscopy. Arrowheads show the yellow overlap of both fluorescence. Bar: 10 μm.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Identification of specific radiolabeled bands in CHO-transfected cells. CHO cells transfected with the (A) NTR1, (B) NTR3, and (C) both receptors were used for photoaffinity labeling with azido-125INT and Western blot analysis by using anti-NTR1 and anti-NTR3 antibodies as described in the Materials and Methods section. n.s., nonspecific.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

6. Fig. 5
Coimmunoprecipitation of the human NTR1 with the human NTR3 in a heterologous expression system. CHO cells transfected either with (A) both receptors or with (B) each receptor independently were incubated with 0.5 nmol/L azido-125INT at 37°C for 40 minutes before ultraviolet irradiation. After immunoprecipitation with the anti-NTR3 antibody, precipitates were analyzed by SDS-PAGE and autoradiography.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Modulation of NT-induced phosphorylation of MAP kinases Erk1/2 by heterodimerization between the NTR1 and the NTR3. CHO cells expressing the indicated receptor or HT29 cells were stimulated with increasing concentrations of NT for 5 minutes at 37°C. (A, B, and C) The phosphorylation of MAP kinases (p-Erk) was determined by immunoblotting by using an antibody directed against the phosphorylated active form of Erk and was representative of a typical experiment. (D and E) Data were standardized from 3 different experiments by using either an anti-Erk total or the anti-NTR3 antibody.
Gastroenterology  , DOI: ( /gast )
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8. Fig. 7
Modulation of the NT-induced PI production by heterodimerization. (A) HT29 cells (■) and CHO cells expressing either the NTR1 (□), the NTR3 (●), or both receptors (○) were stimulated for 15 minutes at 37°C with increasing concentrations of NT. PI accumulation was determined as previously described.24 Values are mean ± SEM from at least 3 independent experiments performed in triplicate. (B) CHO cells were transfected with the human NTR1 alone (0.5 μg of plasmid) (□) or with increasing amounts of plasmid human NTR3: 1 μg (♢), 5 μg (▴), 10 μg (▵). PI accumulation was determined after NT stimulation as described in A. Values are mean ± SEM from 2 independent experiments performed in triplicate.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions