Peroxidase conjugated anti mouse igg

Total and nuclear proteins from tissues and HT29 cells were obtained as previously described [21 (link)]. The expression of several proteins (Table S3) was analyzed by Western blot. Equal amounts of protein were loaded onto SDS/PAGE gels. Membranes were incubated with specific primary antibodies (Table S3) and with a peroxidase-conjugated anti-mouse IgG (Thermo Scientific, Rockford, IL, U.S.A., 1:2500) or anti-rabbit IgG (Thermo Scientific, 1:5000). The signal was detected using supersignal west pico chemiluminescent substrate (Thermo Scientific) and a LAS-3000 (Fujifilm, Barcelona, Spain). The Image Gauge version 4.0 software (Fujifilm) was used to quantify the protein expression by means of densitometry. Total protein data were normalized to β-actin while data of nuclear protein were referred to nucleolin.

Native PAGE was carried out as previously reported [51 (link)]. Briefly, 3–13% acrylamide gradient gels were loaded with 60 μg of mitochondrial protein and processed as described. After electrophoresis, proteins were transferred to a PROTAN^® nitrocellulose membrane (Schleicher and Schuell, Bioscience GmbH, Dassel, Germany) at 35 V, overnight, and probed with specific antibodies raised against the following human OXPHOS subunits: NDUFA9, CORE2, COX2, COX5A, and SDHA (from Mitosciences, Eugene, OR, USA). Peroxidase-conjugated anti-mouse IgG was used as a secondary antibody (Molecular Probes, Thermofisher Scientific, Gilligham, UK). The signal was detected with ECL^® plus (Amersham Biosciences, Freiburg, Germany).