Transfected or infected cells were lysed in an appropriate volume of buffer I (5% SDS, 0.15 M Tris-HCl pH 6.8, 30% glycerol) diluted 1:3 in buffer II (25 mM Tris-HCl pH 8, 50 mM NaCl, 0.5% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with Complete protease inhibitors (Roche Molecular Biochemicals) and 5 mM N-ethylmaleimide (NEM). Lysates were then incubated on ice for 20 minutes and boiled at 95uC for 10 min. Proteins were separated by SDS-PAGE, electroblotted onto a polyvinylidene fluoride membrane (Bio-Rad), and analyzed by western blotting with indicated antibodies (details are given in Supplementary Material and Methods in Text S1).