Protein extraction and phosphatase treatment

Transfected cells and controls were washed in PBS and lysed in ice-cold lysis buffer (50–100 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM DTT, 1% Triton X-100, and mini protease inhibitor cocktail [Roche], with or without phosphatase inhibitor cocktail [Roche]). Insolubilized material was pelleted by centrifugation at 15,000 g. Clarified lysates were incubated with 0.9 mM MnCl₂ and 7 U/µl lysate λPP (New England Biolabs), or 0.9 mM MnCl₂ and H₂O as control, for 1 h at 30°C. The total protein concentration of the lysate was determined by a Bradford protein assay (Bio-Rad). Reactions were stopped, and proteins were denatured in Laemmli buffer for 10 min at 95°C.