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for 48 h in media containing 50 µM Ac
4
ManNAz. Next,
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1
2
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9
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1
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the cells were washed with Dulbecco’s Phosphate-Buffered
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beling buffer (1 % Fetal Bovine Serum (FBS) in DPBS) at
(
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a density of 10x10 cells/mL. The cells were then incubated
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for 1 h on ice. Following cell washing with labeling buffer,
6
they were suspended in labeling buffer at a density of 10x10
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tion experiments, the appropriate ligand in 5 µL of labeling
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(
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0
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0
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0
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0
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reader (PerkinElmer) equipped with a TR-FRET module.
The filter setup consisted of the following: excitation filter
UV2 (TRF), 340 nm; emission filters 495 nm (donor) and
4
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5
20 nm (acceptor); mirror module D400. The measurement
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(
(
(
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Top − Bottom
Y = Bottom +
1 + 10(
log(IC50)−X)∗HillSlope
where ”X” is the logarithm of the competitor concentra-
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librium obtained. ”Top” indicates the maximum amount
of ligand binding without competitor. ”Bottom” expresses
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very high concentration of competitor. ”HillSlope” reflects
the slope factor, which indicates the steepness of the result-
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Acknowledgement We thank Arlene Manelli for provid-
ing the human HRH3-expressing cell line as well as Noah
Pefaur and Marla C. Harris for the human IL-36R anti-
body. We thank Jane Seagal, Sheeba Mathew and Amanda
Horowitz for the mouse IL-36R antibody.
1
65–75.
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DISCLOSURE
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All authors are employees of AbbVie. The design, study
conduct, and financial support for this research were pro-
vided by AbbVie. AbbVie participated in the interpretation
of data, review, and approval of the publication.
(
SUPPORTING INFORMATION AVAILABLE
Experimental procedures and characterization data for all
new compounds available as Supporting Information at
http://pubs.acs.org.
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