Fig. 1. Nucleocytoplasmic and mitochondrial localization of OGT. (A) HeLa cells (top row) and HAEC (bottom row) were grown on glass coverslips, fixed and processed for indirect immunofluorescence using affinity-purified anti-OGT antibodies and a mouse monoclonal antibody raised against cytochrome C. The confocal images representing OGT (green) and cytochrome C (red) were overlayed (merge) to reveal colocalization. Bar, 10 µM. (B) Total cellular lysate (total), nuclear and mitochondrial (mito) fractions from HeLa cells were separated using a 10% PAGE-gel, transferred to nitrocellulose and probed with OGT-specific, affinity-purified antibodies as described in Materials and Methods. Equal amounts of protein were loaded in each lane. Note the enrichment (10-fold) of the 103 kDa species in mitochondria.