Extended Data Fig. 3: VHL interacts with TFAM within mitochondria.
(a) Immunoblot analysis of subcellular fractionation of SK-N-F1 cells. Cell lysates were fractionated into cytosolic and mitochondrial fractions. In addition, aliquots of the mitochondrial fractions were treated with 25 μg/ml Proteinase K with or without treatment with 1% Triton X-100. Fractions were analyzed by western blotting and the localization of VHL or EglN3 was assessed in comparison to that of protein markers of the cytosol (tubulin), outer mitochondrial membrane (TOM20), and mitochondrial matrix (mitochondrial ribosomal protein MRPL37). n = 3 biological independent experiments. (b) Representative images of proximity ligation assay (PLA) signal (green), DAPI (blue) and MitoTracker Red (red) triple staining in 786-0 cells expressing VHL wildtype. The images show the maximal intensity projection of the signal/staining. (c) 3D rendering and (d) Orthogonal view showing co-localization of PLA signal in mitochondria (yellow). Magnification 63x; scale bar: 5 µm. (b-d) Similar results were seen more than three times.
