Fig. 3.

Human GRPELs are homodimers with nearly identical proximal proteins A. Design of proteomic experiment using BioID assay. 143B cells were transiently transfected with indicated constructs. Biotin was added to the culture media for 24 h. The cells were lysed and biotinylated proteins were enriched by Strep-Tactin®. The samples were analyzed by mass spectrometry. B. Western blot analysis of GRPEL1-BirA* or GRPEL2-Bir* expression. GAPDH was used as a loading control. C. Intracellular localization of GRPEL1-BirA* or GRPEL2-Bir* determined by immunocytochemistry. MitoVector was used to label mitochondria, DAPI shows nuclei. Scale bar, 10 µm. D. Mitochondrial specificity of GRPEL1 or GRPEL2 proximal candidates before filtering. E. Heat map of the unique proximal proteins of GRPEL1 or/and GRPEL2. The spectral counts for mtHSP70 (HSPA9) are also shown. F. Clear native gel electrophoresis analysis of GRPELs in HEK293 cells G. Blue native gel electrophoresis analysis of GRPELs in HEK293 cells. H. Reducing and non-reducing western blotting of GRPELs. GAPDH was used as a loading control. I. Two-dimensional non-reducing/reducing SDS-PAGE of GRPELs. Red arrows point to the monomer in second dimension.