Immunoblot analysis. Cells were lysed with M-PER Protein Extraction Reagent (Pierce) supplemented with a protease inhibitor mixture. A bicinchoninic acid assay (Pierce) was used to measure the protein concentrations in the extracts. For immunoblot analysis, immunoprecipitates or whole-cell lysates were loaded and subjected to SDS–PAGE. The proteins in the gel were transferred onto nitrocellulose membranes and then blotted as described previously50–52.