1868. |
β-arrestin- but not G protein-mediated signaling
by the “decoy” receptor CXCR7
Sudarshan Rajagopal, Jihee Kim, Seungkirl Ahn, Stewart Craig, Christopher M. Lam, Norma P. Gerard, Craig Gerard, and Robert J. Lefkowitz,
Duke University Medical Center,
PNAS,
107(1868),
(2010)
Link To Paper
Keywords:
Biased receptor, Chemokine, G protein-coupled receptors, Seven-transmembrane receptors Materials & Methods:
Confocal Microscopy. HEK-293 cells were split into 35-mm glass-bottom dishes (MatTek) and transfected with 1 μg of CXCR7 and 0.2 μg of β-arrestin-2-GFP. Forty-eight hours after transfection, cells were starved for at least 5 h in serum-free media prior to treatment with ligand. For live cell imaging, cells were maintained at 37 °C with a heating plate while confocal microscopy was performed. SDF-1α and ITAC were added to a final concentration of 100 nM and images were taken from 0 to 30 min. For immunostaining, cells were treated for 30 min with 100 nM ligand, followed by aspiration of serum and fixation. Samples were then washed followed by permeabilization and blocking. Cells were then incubated overnight with primary antibody and then washed prior to incubation with secondary antibody at 1∶500 dilution. Samples were then washed and visualized. Microscopic Technique
Confocal Microscopy Cell Type(s)
HEK-293 cells |
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35-mm glass-bottom dishes (MatTek) 