Cells were harvested in IP buffer [50 mM tris-HCl (pH 8.0), 1% NP-40, and 150 mM NaCl] supplemented with protease inhibitors, phosphatase inhibitors, 1 mM PMSF, 20 mM N-ethylmaleimide (Sigma-Aldrich), and 10 μM MG-132 (Sigma-Aldrich). Lysates were cleared by centrifugation (15,000g for 20 min at 4°C) and incubated for 3 hours or overnight at 4°C with indicated antibodies (1 to 2 μg each). Protein G–magnetic beads were added for 1 hour. Beads were washed five times in IP buffer and resuspended in Laemmli loading buffer for immunoblotting.

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