Western blot analysis was done as described previously41. Briefly, cells were lysed in a lysis buffer (20 mM Tris-HCl, pH 7.4, 2% Triton X-100, 10 mM EDTA, 5 mM NaF and 1 mM sodium orthovanadate). Protein concentration was determined by BCA assay (Pierce, Rockford, IL). Then proteins were resolved in 12% SDS polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were then incubated overnight at 4 °C in blocking solution containing 5% nonfat dry milk in PBS with 0.1% Tween-20. Subsequently the membranes were incubated with primary antibodies, followed by incubation with HRP (horseradish peroxidase)-conjugated second antibodies. Detection of HRP was performed by SuperSignal West Pico Chemiluminescent Substrate (Pierce). Antibodies are listed in Supplementary Table 3.
