Fig. 3. O-GlcNAc-modified glycoproteins do not accumulate in mitochondria although mOGT is catalytically active. (A) Untransfected HeLa cells (upper row) were grown on glass coverslips, fixed and processed for indirect immunofluorescence to reveal endogenous OGT (OGT) and O-GlcNAc (RL2) localization. The two images were overlaid to determine colocalization (Merge). (B) HeLa subcellular fractions were probed for the O-GlcNAc-modified proteins. Equal amounts of total HeLa lysate (total), cytosol (cyto), nuclear (nuc) and mitochondrial proteins were separated using a 10% Bis-Tris SDS gel, transferred to nitrocellulose and probed with the RL2 monoclonal antibody. The migration of molecular weight markers (kDa) is shown on the left. (C) To demonstrate that mOGT is catalytically active when mislocalized from mitochondria to the cytoplasm, HeLa cells were transfected with GFP-mOGT for 24 hours, fixed and processed to visualize GFP-mOGT. Indirect immunofluorescence was used to localize O-GlcNAc (RL2). Bars, 10 µM.