Both authors contributed equally to the work.
Biochemical and pharmacological characterization of nuclear urotensin-II binding sites in rat heart
Article first published online: 10 APR 2012
© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society
British Journal of Pharmacology
Special Issue: Themed Section: Secretin Family (Class B) G Protein-Coupled Receptors - from Molecular to Clinical Perspectives. Guest Editors: David R Poyner and Debbie L Hay
Volume 166, Issue 1, pages 243–257, May 2012
How to Cite
Doan, N., Nguyen, T., Létourneau, M., Turcotte, K., Fournier, A. and Chatenet, D. (2012), Biochemical and pharmacological characterization of nuclear urotensin-II binding sites in rat heart. British Journal of Pharmacology, 166: 243–257. doi: 10.1111/j.1476-5381.2011.01710.x
- Issue published online: 10 APR 2012
- Article first published online: 10 APR 2012
- Accepted manuscript online: 1 NOV 2011 06:47AM EST
- Received; 21 March 2011; Revised; 12 August 2011; Accepted; 17 September 2011
Figure S1 (A) Amino acid sequence of synthesized peptides. (B) Analytical data obtained by MALDI-TOF spectrometry and by RP-HPLC. aPercentage of purity determined by HPLC using a buffer system [A = H2O (pH 2.5) and B = 100% CH3CN] with a gradient slope of 1% B per minute, at a flow rate of 1 mL·min−1 on a Vydac C18 column (5 μm particle size, 300 Å pore size). Detection at 214 nm. bObserved m/z-value compared with the calculated [M + H]+ monoisotopic mass. (C) Effect of hU-II or N-biotin-[Ahx0, Bpa3]-hU-II on Ca2+ mobilization using CHO cells co-expressing human UT receptors and a mitochondrial apo-aequorin protein. Data are expressed as a percentage of the response (bioluminescence) obtained with digitonin (50 μM) added to the same 96-well culture plate. Data represent the mean ± SEM of at least four independent assays performed in duplicate.
Figure S2 Enlargement of a nucleus from Figure 4D. Nucleus was stained with DRAQ5TM (blue colour). Left ventricular sections are double-stained for lamin A (red colour) and UT (green colour). This enlargement demonstrates the co-localization of UT (yellow colour, arrowhead) and lamin A.
Figure S3 Distribution of FITC-conjugated hU-II and URP in fixed untransfected CHO-K1 cells. Nuclei were stained with PI.
Figure S4 Distribution of FITC-conjugated hUII and URP in living (A) HeLa and (B) HEK-293 cells. Nuclei were stained with DRAQ5TM.
Figure S5 Internalization of 125I-hU-II and 125I-URP in CHO-K1 cells. CHO-K1 cells in 12-well plates were incubated for various periods of time in FBS-free media containing 0.2 nM 125I-hU-II or 125I-URP. Internalization was stopped by washing, and cells were then solubilized with 1 M NaOH, and the radioactivity was quantified using a γ-counter. Membrane-bound radioactivity was evaluated by incubation of CHO-K1 cells with 125I-labelled peptide (0.05 nM) for 30 s.
Appendix S1 Methods.
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