For construction method of vectors encoding C-terminal Cerulean (Addgene, 27795) or Venus (Addgene, 27793) fluorescent protein-tagged versions of GPR35-T108T, GPR35-T108M, ATP1A1, CCR5, and CXCR2, see the “Cloning and constructs” section. Twenty-four hours before transfection, HEK293T cells were seeded onto poly-l-lysine–coated glass coverslips in six-well plates at a density of 0.5 × 106 per well. Plasmid DNA (2500 ng) was transfected using Lipofectamine 2000 (Thermo Fisher, 11668027) in Opti-MEM Reduced Serum Medium (Thermo Fisher 31985062) according to the manufacturer’s protocol. Pairs of different constructs were mixed 1:1 before transfection. Twenty-four hours after transfection, cells were washed with PBS and fixed with 4% paraformaldehyde in 0.12 M sucrose for 15 min, followed by three further PBS washes. Coverslips were then lifted and mounted onto microscope slides. FRET measurements were performed using a Leica TCS SP5 confocal microscope (Leica Biosystems) and Leica Application Suite Advanced Fluorescence software. FRET measurements were taken using the acceptor photobleaching method. Briefly, cells expressing similar fluorescence levels of both transfected constructs were identified. Baseline Cerulean “donor” emission was measured during excitation at 405-nm wavelength at 10% laser power. The Venus “acceptor” protein was then photobleached using 20 pulses of 514-nm wavelength at 98% laser power so that it was no longer able to accept energy from the Cerulean donor. The Cerulean emission during excitation at 405-nm wavelength at 10% laser power was then remeasured. FRET efficiency was then calculated from pre- and post-photobleaching emission values. Measurements from nonbleached cells within the same field were used to normalize.

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