Cells were lysed in 1% Triton buffer [25 mM tris (pH 7.4), 100 mM NaCl, 10% glycerol, 1 mM EDTA, and 1% Triton-X 100], supplemented with protease inhibitors (Roche) and phosphatase inhibitors (Sigma-Aldrich) for 10 min, with brief vortexing. Lysates were clarified by centrifugation at 18,000g for 15 min at 4°C, and protein concentrations were measured using Bradford reagent (Bio-Rad). Standard immunoblotting procedures were performed as we described previously (14).

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