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. 2015 Apr 27;112(19):6050–6055. doi: 10.1073/pnas.1424017112

Fig. 2.

Fig. 2.

Interaction of mitochondrial complex IV with the putative short OGT isoform within normal and diabetic cardiac mitochondria and the corresponding complex IV activity assay. (A) BN PAGE analysis of purified heart mitochondria samples followed by second dimensional SDS gel immunoblotted with anti-OGT antibody. (B) Immunoprecipitation of complex IV from normal and diabetic mitochondria with anti-complex IV antibody labeled beads (Abcam) and immunoblotted against OGT and complex IV antibody. (C) Quantification of immunoprecipitated OGT/complex IV levels (n = 3). (D) CTD blot on complex IV immunoprecipitated sample showing changes in O-GlcNAcylation pattern of complex IV subunits between normal and diabetic samples. Arrows indicate bands that show altered O-GlcNAc labeling. (E) OGT activity on immunoprecipitated complex IV beads with and without external substrate peptide (NH, normal and DH, diabetic samples). (F) Complex IV activity assay as analyzed by following oxidation of cytochrome c resulting in loss of absorbance at 550 nm. (G) Rate of cytochrome c oxidation from normal and diabetic samples obtained by the initial slope from F above (n = 3). (*P < 0.05).