Culturing HEK-293 and MEF Cells

HEK-293 cells stably expressing the M₃ muscarinic acetylcholine receptor were cultured in Dulbecco’s modified Eagle’s medium (DMEM/F12) supplemented with 10% fetal calf serum, 100 U/mL penicillin G, and 0.1 mg/mL streptomycin as described [23 (link)]. Stock cultures of HEK-293 cells were grown in the presence of 0.5 mg/mL G418. Mouse embryonic fibroblasts (MEFs) from CIB1-deficient mice were made by J.L. Black in the laboratory of Dr. Leslie V. Parise (University of North Carolina at Chapel Hill, NC, USA) [36 (link),37 (link)]. These MEFs, along with MEFs from Gα_q/11 double-deficient mice [38 (link)], were cultured in DMEM/F12 medium with 10% fetal calf serum, 100 U/mL penicillin G, and 0.1 mg/mL streptomycin. Transfection of HEK-293 cells was performed with Lipofectamine 2000 (Invitrogen GmbH, Karlsruhe, Germany), while MEFs were transfected with Turbofect (Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s instructions. For microscopy, the cells were seeded onto poly-l-lysine-coated 8-well slides (μ-slide; ibidi GmbH, Martinsried, Germany). Before experiments, the cells were kept in serum-free medium overnight.