Total proteins were isolated from cell pellets by extraction with ice-cold 20 mM Tris, pH 7.4, 1% Triton X-100 (membrane-research grade, Roche), plus protease inhibitors (Complete mini tablet, 0.7 μg/ml pepstatin A, 2 μg/ml aprotinin, 5 μg/ml leupeptin [Roche]). Following homogenization through a 25-gauge needle (~10 passes), extracts were centrifuged at 1000 × g for 10 minutes, at 4°C, to remove debris. Typically, 20–40 μg of protein (determined by Bio-rad Dc Protein Assay) was separated by SDS-PAGE, for subsequent immunoblotting, as described [12]. 16.5% tris-tricine SDS-PAGE gels were used for subunit c immunoblotting experiments.