Western Blot Analysis of Cellular Proteins

Cells were lysed in SDS sample buffer (62.5 mM Tris–HCl pH 6.8, 2% sodium dodecyl sulfate) supplemented with protease, phosphatase and proteasome inhibitors, pre-heated at 95 °C^{40 (link)}. Thereafter, samples were sonicated and protein amount dosed with Pierce BCA protein Assay Kit (Thermo Fisher Scientific). Sixty µg of protein extracts were then separated on NuPage 4–12% Bis–Tris Gels (Thermo Fisher Scientific) and transferred with iBlot System (Thermo Fisher Scientific). Membranes were incubated with blocking buffer (5% nonfat dry milk in TBS-T solution) for 1 h at room temperature and probed overnight at 4 °C with appropriate primary antibodies diluted 1:1000 in blocking buffer: antiFAN83B (Atlas Antiboides), anti-vimentin and anti-SDHA (Abcam), anti-p-ERK1/2, anti-ERK1/2, anti-P-AKT and anti-AKT (Cell Signalling), as previously described^{40 (link)}. After washing, membranes were incubated for 1 h at room temperature in the presence of the appropriated HRP-conjugated secondary antibody (Merck Millipore, Burlington, Massachusetts, USA) diluted to 1:5000 in blocking buffer. Detection was performed utilizing Westar Supernova ECL Substrate (Cyanagen, Bologna, Italy) and images acquired with c400 camera (Azure Biosystems, Sierra Ct, Dublin, USA). Band intensity was quantified with FIJI software^{41 (link)}. Full-length blot images are shown in Supplementary Figs. 4–8.