All experimental procedures were carried out at +4°C. Tissue extracts were prepared by a modification [24] of the protocol described elsewhere [38], [39]; powdered tissue preparations (25–35 mg) were homogenized in 4 volumes of Buffer C (20 mM HEPES, pH 7.9, 0.42 M NaCl, 25% glycerol, 1.5 mM MgCl2, 0.4 mM EDTA, 0.5 mM DTT, 0.2% NP-40, 25 µM proteasome inhibitor MG132 and 5×protease inhibitors (Complete, Roche, Switzerland)) by 10 strokes with plastic pestles (Sigma-Aldrich, St. Louis, MO, USA) in Eppendorf tubes, incubated for 10 min, homogenized again by 10 strokes and centrifuged at 20,000×g for 15 min at 4°C. The supernatant was kept at −80°C until assayed. Protein concentration was determined using the DC protein assay.
