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. 2018 Jan 31;8(1):170207. doi: 10.1098/rsob.170207

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© 2018 The Authors.

Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.

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Figure 2. — Recombinant ACKR3 and AVPR1A form heteromeric complexes. (a) Typical PLA images for the detection of individual receptors and receptor–receptor interactions in HEK293T cells transfected with DNA encoding HA- or FLAG-tagged receptors. Ctrl: cells transfected with pcDNA. Images show merged PLA/DAPI signals acquired from z-stack images (n = 10; thickness 1 µm, bottom to top). Scale bars, 10 µm. (b) Quantification of PLA signals per cell as in (a). n = 3 independent experiments with n = 10 images per condition and experiment. (c) HEK293T cells expressing HA-AVPR1A and FLAG-ACKR3 were lysed (input), the lysate was immunoprecipitated (IP) with anti-HA, followed by immunoblotting (IB) to detect HA-AVPR1A (left) and FLAG-ACKR3 (right) in the IP samples. IP control: precipitate after incubation of cell lysates with IgG-coupled resin. (d,e) Intermolecular BRET assays. Cells were co-transfected with AVPR1A-hRluc plus EYFP (open circles) or ACKR3-EYFP (grey squares) at various acceptor : donor ratios (d) and with increasing amounts of AVPR1A-hRluc and ACKR3-EYFP at a constant ratio of 1 : 10 (e).