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Receptor binding experiments
Receptor binding data were determined according to protocols as
described previously.33–35 In detail, hNTR1 binding was measured
using homogenates of membranes from Chinese hamster ovary
(CHO) cells stably expressing the human NTR1 receptor at a final
concentration of 2–4 mg/well and the radioligand [3H]neurotensin
(specific activity 116 Ci mmol-1; Perkin Elmer, Boston, MA) at a
concentration of 0.5 nM.34 Specific binding of the radioligand was
determined at a KD value of 0.47–0.54 nM and a Bmax of 5050–7025
fmol mg-1 protein. NTS2 binding was done using homogenates
of membranes from human embryonic kidney cells (HEK 293),
which were transiently transfected with the pcDNA3.1 vector con-
taining the human NTS2 gene (UMR) by the calcium phosphate
method. Membranes were incubated at a final concentration of
20 mg/well together with 0.5 nM of [3H]NT(8–13) (specific activity
136 Ci mmol-1; custom synthesis by GE Healthcare, Freiburg,
Germany) at a KD value of 0.86–1.14 nM and a Bmax of 665–
1380 fmol mg-1 protein.33 Specific binding of the radioligand
was determined at a KD value of 0.47–0.54 nM and a Bmax of
5050–7025 fmol mg-1 protein. Unspecific binding was determined
in the presence of 10 mM neurotensin (for NTS1) or NT(8–13)
(for NTS2) and the protein concentration was established by the
method of Lowry using bovine serum albumin as standard.36 Data
analysis of the resulting competition curves was accomplished
by non-linear regression analysis using the algorithms in PRISM
(GraphPad Software, San Diego, CA). EC50 values derived from
the resulting dose response curves were transformed into the
corresponding Ki values utilizing the equation of Cheng and
Prusoff.37
Acknowledgements
The generous financial support of our research by the Deutsche
Forschungsgemeinschaft (DFG) and the Friedrich-Alexander-
Universita¨t Erlangen-Nu¨rnberg (FAU) is gratefully acknowl-
edged. We would also like to thank Iris Torres for technical
assistance.
13 J. P. Vincent, J. Mazella and P. Kitabgi, Top. Prot. Sci., 1999, 20, 302.
14 P. R. Dobner, Peptides, 2006, 27, 2405.
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