Cell culture grade dmso

Assay buffer consisted of MilliQ water, 135 mM NaCl (AppliChem, Darmstadt, Germany), 1 mM CaCl₂ (AppliChem), 5 mM KCl (AppliChem), 1 mM MgCl₂ (AppliChem), 11 mM Na-HEPES (pH = 7.4) (Sigma-Aldrich, Taufkirchen, Germany), protease inhibitor cocktail (according to the manufacturer's description, Roche, Basel, Switzerland) and 0.1% Pluronic F-127 (Sigma-Aldrich).
Muscarinic acetylcholine receptor ligands acetylcholine, arecoline, pirenzepine, pilocarpine, atropine and scopolamine were purchased from Sigma-Aldrich and carbachol from Tocris Bioscience (Abingdon, UK). The syntheses of the fluorescent ligands UR-MK342 and UR-CG072 [16 (link)] and the dualsteric M₂R ligands UR-SK59 [50 (link)], UR-SK75 [50 (link)] and UNSW-MK259 [51 (link)], showing also high M₄R affinity, were described previously. All ligand stock solutions were prepared using cell culture grade DMSO (AppliChem) and stored at −20°C.

Cells were starved in DMEM without serum overnight in the presence of either 500 nM GDC‐0941 (Selleckchem) or an equal volume of cell culture grade DMSO (AppliChem). Cells were stimulated with 20 μg/ml insulin (Sigma) in DMEM or control treated with equal amounts of DMEM for 1 min prior to fixation in PIP₃‐Immobilisation fixative and stained with for PIP₃ (anti‐PIP₃, Echelon) and F‐actin (Phalloidin, Thermo Fisher Scientific).