HEK293T cells were transfected with plasmids encoding muscarinic receptor M3, PLCß3-YFP, and the G protein subunits Gβ1-, Gɣ2-, and either cyan fluorescent protein (CFP)–Gαq or CFP-GαqQ209L and then were seeded for 24 hours in six-well plates. FRET measurements were performed 48 hours after transfection. The cells were incubated in FRET buffer [137 mM NaCl, 5.4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 10 mM Hepes (pH 7.3)], and FRET imaging was performed with a light-emitting diode excitation system on the fluorescence microscope Eclipse Ti by Nikon. Emitted light was measured at 425 and 500 nm in an interval of 2 s. For the experimental setup, cells were exposed to a constant flow of FRET buffer in the ALA-VC3-8SP perfusion system (ALA Scientific Instruments). The buffer was replaced with buffer supplemented with 10 μM CCh for 30 s, which was followed by a washing step of 1.5 min. Thereafter, FRET buffer including CCh was re-added for another 30 s before the buffer was again replaced by a mixture of buffer with 10 μM CCh and 1 μM FR. After the cells were treated with FR, a second washing step of 1 min with buffer flow was implemented before the cells were once more stimulated with CCh.

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