Dry transfer apparatus

Complex I assembly was analyzed by Blue Native electrophoresis (BN Page) according to [24 (link)]. Briefly, mitochondria were isolated from cells pellets by differential centrifugation (10,000× g, 10 min, 4 °C) after digitonin permeabilization. Mitochondrial complexes were solubilized in Laurylmaltoside (3 g/g of proteins), and 50 µg of proteins were loaded and resolved in a native 4–16% acrylamide gel. Proteins were transferred to the PVDF membrane in a dry transfer apparatus (Bio-Rad, Marnes-La-Coquette, France). Membranes were immunoblotted with antibodies raised against NDUFB6 and NDUFS2 for complex 1, SDHA for complex 2 (used as loading reference), III core 2 for complex 3, and COX Va for complex 4 (1/1000e, Abcam, Cambridge, UK), and bands were revealed by chemiluminescence on an Odyssey apparatus (Li-Cor Biosciences). For supercomplexes detection, the same protocol was applied, except to larylmatoside solubilization, which was replaced with digitonin (3g/g of proteins).

Immunoprecipitation and western blotting were performed as described previously42 (link)43 (link). MCF-7 and MDA-MB-231 cells were harvested in 4 °C phosphate-buffered saline and cell pellets were lysed with RIPA lysis buffer (Millipore, Bedford, MA, USA) for 30 min on ice. Cell lysis supernatant liquid was obtained by centrifugation at 10,000 × g for 10 min, incubated with protein-G beads (Roche, Indianapolis, IN) and anti-β-catenin, and subjected to western blotting. For the western blotting assay, cellular extract proteins were separated by SDS-polyacrylamide gel (SDS-PAGE) and transferred to nitrocellulose membrane (Millipore) using a dry transfer apparatus (Bio-Rad). After blocking nonspecific binding with 5% milk buffer, the membrane was incubated with primary antibodies: anti-TAT (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-LEF-1(Epitomics, Burlingame, CA, USA) and anti-β-catenin (Epitomics, Burlingame, CA, USA). The proteins were visualized using ECL (Amersham Pharmacia Biotech) and coupled using the Bio-Rad Chemiluminescent Detection System.