Cox2p

For protein preparations, yeast cultures were grown overnight in 20 ml YPD and crude cytoplasmic and mitochondrial fractions were prepared as described previously [45] (link). Protein concentration (A₂₆₀) was measured by NanoDrop (Thermo Fisher, Waltham, MA). Preparation of 15% SDS polyacrylamide gels and protein electrophoresis were carried out according to Laemmli [46] (link). For Western blot analysis, proteins were transferred onto a PVDF membrane (Millipore, Billerica, MA), probed with primary antibodies and detected with HRP-conjugated secondary antibodies using the ECL Prime Kit (GE Healthcare, Little Chalfont, UK). Primary antibodies were directed against HA (Roche, Basel, Switzerland) and Cox2p (Invitrogen, Carlsbad, CA), respectively.

Preparation of 10% SDS polyacrylamide gels and protein electrophoresis were carried out according to Laemmli [25] (link). For Western blot analysis proteins were transferred onto a PVDF membrane (Millipore, Billerica, MA), probed with primary antibodies and detected with HRP-conjugated secondary antibodies and the ECL-Plus Kit (GE Healthcare, Little Chalfont, UK). Primary antibodies were directed against cMyc (Roche), Cox2p (cytochrome c oxidase subunit 2; Invitrogen, Carlsbad, CA), Aco1p (aconitase; kind gift of R. Lill), Pgk1p (phosphoglycerate kinase; Invitrogen), Dpm1p (dolichol phosphate mannose synthase; Invitrogen), Cit1p (citrate synthase; kind gift of T. Fox), Ccp1p (cytochrome c oxidase; kind gift of W. Neupert), Tom40p (subunit of the outer membrane translocase; kind gift of J. Brix) and lipoic acid (LA; Calbiochem).