Complete protease inhibitor cocktail and anti phosphatases

Tissue samples were lysed in ice‐cold RIPA buffer (50 mM Tris‐HCl, pH 8, 12 mM deoxycholic acid, 150 mM NaCl and 1% NP40, supplemented with Complete Protease Inhibitor Cocktail and anti‐phosphatases, Roche) using a Teflon‐on‐glass homogenizer and then centrifuged at 7000 g for 10 min at 4°C. The protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Samples (30 μg) were boiled for 5 min in Laemmli's buffer and run on Bis‐Tris gels with a Bolt^TM mini gel tank (Invitrogen) using MOPS SDS running buffer Bolt^TM (Invitrogen). After electrophoresis, proteins were transferred to nitrocellulose membranes using an iBlot^®2 gel transfer device (Invitrogen) for blotting with appropriate antibodies. Proteins were visualized with an enhanced chemiluminescence western blot detection system (GE Healthcare Bio‐Sciences AB), after exposure to CL‐XPosure Film (Thermo Scientific).