Immunoblotting was performed as previously described [19 (link)]. The adherent cells were scraped from the dish using a cold plastic cell scraper, and the cell suspension was transferred into an Eppendorf tube. Cell samples were sonicated in a lysis buffer. Whole-cell lysates were centrifuged in a microcentrifuge at 13,000× g for 20 min at 4 °C. The tube was then removed from the centrifuge, and placed on ice. Subsequently, the supernatant was aspirated, placed in a new tube kept on ice, and the pellet was discarded. Lysed samples were mixed with sample buffer and separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The separated proteins were transferred to a nitrocellulose membrane and blotted with the appropriate primary and secondary antibodies. Protein bands were detected using enhanced chemiluminescence (GE Healthcare, RPN2109, Hatfield, UK).
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