Cells were lysed with Nonidet P-40 (NP-40) buffer (20 mM Tris [pH 8.0], 137 mM NaCl, 1% NP-40, 2 mM EDTA) or RIPA buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% NP40, 0.1% SDS, 0.25% sodium deoxycholate, 1 mM EDTA) supplemented with protease and phosphatase inhibitors. Lysates were cleared by centrifugation for 10 minutes at 14,000 rpm and 4°C. Immunoprecipitation was performed at 4 °C with the indicated antibodies, and the products were collected on Dynabeads® Protein A or G (Life Technologies) as described previously (26, 27). For Western blotting, proteins in whole cell lysates (WCL) were resolved by SDS-PAGE and transferred to PVDF membranes (Immobilon; Millipore). The membranes were blocked in 5% BSA or 5% nonfat dried milk in PBST (PBS + 0.1% Tween-20). The images presented are representative of three independent experiments. The relative integrated density of each protein band was digitized by NIH image J.

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