Enhanced chemiluminescence

Cells were lysed in ice-cold buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Igepal and 1 mM EDTA with protease inhibitor cocktail (539137, Millipore). Samples were separated by SDS-PAGE followed by transfer to nitrocellulose membranes (Cyvita). Membranes were blocked in 5% milk in tris-buffered saline with 0.1% Tween-20 (TBST). Primary antibodies diluted in 5% milk-TBST were incubated with the membrane overnight at 4°C. Membranes were washed in TBST, incubated with secondary antibodies for 1 h, washed with TBST and detected by enhanced chemiluminescence (Promega). For Fig. 7, blots were quantified using Image Studio Lite (LI-COR). Band intensity was measured and normalised to GAPDH for each sample.

Frozen myocardial tissue was homogenized with the Radio-Immunoprecipitation Assay lysis buffer [50-mM Tris–HCl pH8, 150-mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1% sodium dodecyl sulphate (SDS), 1-mM phenylmethylsulfonyl fluoride, 10-mg/ml aprotinin, 10-mg/ml leupeptin, and 10 mg/ml pepstatin] and centrifuged at 14000 g for 10 min at 4°C. The protein concentration was measured by protein assay (Bio-rad Laboratories s.r.l, Hercules, CA, USA). Equal amount of protein (40 μg) was separated by 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) or precast 4–20% Bolt™ Mini Gels (Invitrogen ™, Life Technologies Italia, MB, Italy) and transferred to a polyvinylidene fluoride (PVDF) membrane. The following primary antibodies were used: antimouse monoclonal PGC-1α (Calbiochem, Millipore, MA, USA); antirabbit monoclonal ERRα (Abcam, Cambridge, UK); antirabbit polyclonal TFAM (ATLAS antibodies AB, AlbaNova University Center, Stockholm, Sweden); antimouse β-Actin (− Aldrich, St-Louis, MO, USA). Primary antibodies were visualized using horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, Laboratories, Inc., USA). Signals were detected by enhanced chemiluminescence (Promega corporation, Madison, WI, USA).

IPEC-J2 cells were lysed for 30 min with radioimmunoprecipitation assay lysis buffer containing PMSF and then centrifuged at 12, 000g for 15 min. The proteins in the supernatant were separated by 12% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore). The membrane was blocked at room temperature (RT) for 2 h with 5% nonfat milk and then incubated at 4 °C for 8 h with primary antibodies, followed by incubation with a secondary antibody at RT for 2 h. Signals were detected by enhanced chemiluminescence (Promega).