Western Blot Analysis of Protein Expression

Western blot analysis was performed as previously described [50 (link)]. Briefly, cells were sonicated on ice for 10 min in buffer supplemented with a protease and phosphatase inhibitor cocktail (Pierce) as described [13 (link)]. An amount of 60 µg was separated on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis SDS–PAGE. Proteins were transferred on a nitrocellulose membrane and incubated with primary antibodies (Table Supplementary 3, online resource) overnight at 4 °C. Blots were then incubated in secondary antibodies (polyclonal goat anti-rabbit HRP 1:1000, Dako or 1:2700, Life Technologies) and the immune complexes were revealed with a chemiluminescence assay (Amersham). The nitrocellulose membrane was stripped and re-probed with an anti-actin antibody (1:1000) as the loading control.