Vibracell 75 186

For dephosphorylation experiments, cells and hippocampi were homogenized in a buffer containing 100 mM NaCl and 50 mM Tris-Cl at pH 7.4 with 1% (v/v) IGEPAL^® CA-630 (ThermoFisher, Saint-Herblain, France) and a protease inhibitors cocktail without EDTA (Roche, Neuilly sur Seine, France) using either a “Precellys 24” (Bertin technologies, St Quentin-en-Yvelines, France) tissue homogenizer and followed by sonication with “vibracell 75 186” device (Sonics, Newton, CT, USA). Homogenates were centrifuged at 16,100 g for 20 min at 4°C with an Eppendorf 5415R centrifuge (Eppendorf, Hamburg, Germany), sonicated for 10 s and protein amounts normalized following a bicinchoninic acid protein assay (ThermoFisher). Samples were diluted to 1.0 mg/ml protein using homogenization buffer and incubated with 20 U/μl lambda phosphatase in MnCl₂ and enzyme buffer as supplied with the lambda protein phosphatase kit (New England Biolabs, Evry-Courcouronnes, France) for 3 h at 30°C. The reaction was stopped by the addition of sample buffer (Life Technologies) and heating to 95°C for 5 min. Control samples were treated identically without the addition of lambda phosphatase.

For dephosphorylation experiments, cells or tissues were homogenised in a buffer containing 100 mM NaCl and 50 mM Tris-Cl at pH 7.4 with 1% (v/v) IGEPAL® CA-630 and a protease inhibitors cocktail without EDTA (Roche, Neuilly sur Seine, France) using either a “Precellys 24” (Bertin technologies, St Quentin-en-Yvelines, France) or a Tissue Master 125 (Omni International, Kennesaw, GA, USA) tissue homogenizer and followed by sonication with “vibracell 75 186” device (Sonics, Newton CT, USA). Homogenates were centrifuged at 16,300 g for 20 min at 4 °C with an Eppendorf 5415R centrifuge (Eppendorf, Hamburg, Germany), sonicated for 10 s and protein amounts normalized following a BCA protein assay (ThermoFisher, Waltham, MA, USA). Samples were diluted to 1.0 mg/mL protein using homogenisation buffer and incubated with 20 U/μL lambda phosphatase in MnCl₂ and enzyme buffer as supplied with the lambda protein phosphatase kit (New England Biolabs, Ipswich, MA, USA) for 3 h at 30 °C. The reaction was stopped by the addition of sample buffer (National Diagnostics, Hull, UK or Life Technologies, Courtaboeuf, France) and heating to 95 °C for 5 min. Control samples were treated identically without the addition of lambda phosphatase.

Muscular tissues were lysed in RIPA lysis buffer (MILLIPORE, Burlington, MA, USA) with sodium orthovanadate (SIGMA-ALDRICH), phosphatase inhibitors (Phosphatase inhibitor cocktail 3; ROCHE, Boulogne-Billancourt, France) and protease inhibitors (Complete; ROCHE) using a Precellys 24 tissue homogeniser (BERTIN TECHNOLOGIES, Aix-en-Provence, France), followed by sonication with a Vibracell 75186 (SONICS, Newton, CT, USA). Total protein levels were quantified using a bicinchoninic acid protein assay kit (THERMOFISHER, Waltham, MA, USA). Acetylcholine concentration was determined in tissue homogenates containing equal amounts of proteins (75 µg) (Amplex red acetylcholine/acetylcholinesterase assay kit; THERMOFISHER).