Only peptide identifications having a >95% probability mainly because determined by the PeptideProphet algorithm (51) were accepted mainly because valid. separate experiments) from mitochondria enriched from bovine heart cells, (ii) enzymatic labeling of bovine heart mitochondria with UDP-azido-GalNAc via the mutant galactosyltransferase GalT1(Y289L), and (iii) azido-GalNAc metabolic labeling coupled with click chemistry and streptavidin enrichment to capture glycoproteins from mitochondria enriched from rat neuroblastoma B103 cells. Collectively, these studies yielded 84 glycoprotein candidates with known mitochondrial function (supplemental Table 1). We note that LC-MS/MS offered only protein identifications of mitochondrial proteins that were enriched via one of the aforementioned glycosylation-enrichment approaches. Consequently, their status as (20) (supplemental Table 1). Among these glycoprotein candidates, we observed two mitochondrially encoded proteins: cytochrome oxidase subunit 2, which is a novel statement, and NADH-ubiquinone oxidoreductase chain 4, which was recently recognized by Ma (20) as with in Fig. 1< 0.0001 NT siRNA; NS, not significant NT siRNA; #, < 0.05 mOGT siRNAs (1 and 2); = 3 experiments, one-way ANOVA, Bonferroni-corrected Tukey's test). and and < 0.05 NT siRNA; NS, not significant NT siRNA; ***, < 0.001 NT siRNA; #, < 0.05 mOGT siRNAs (siRNAs 1 and 2); data points S.E., = 7C9 Western blots, one-way ANOVA, Bonferroni-corrected Tukey's test). < 0.05 NT siRNA; #, < 0.05 mOGT siRNAs (siRNAs 1 and 2), = 8C27 cells/condition from two experiments, one-way ANOVA, Bonferroni-corrected Tukey's test). < 0.05 NT siRNA, = 15C30 cells/condition, one-way PHA-680632 ANOVA, Bonferroni-corrected Tukey's test). Next, we tested the ability of siRNAs to reduce the protein levels of the OGT isoforms by European blotting (Fig. 1, and and and and was compiled from two self-employed experiments with S.E. (= 9C20 cells). For pub graphs shown in and = 100C150 cells/condition), one-way ANOVA, Bonferroni correction for multiple comparisons. *, < 0.05; **, < 0.01; ***, < 0.001 NT siRNA. In contrast, ncOGT siRNAs significantly reduced the protein levels of both the ncOGT PHA-680632 and mOGT bands (shown for one representative ncOGT siRNA in Fig. 1 (and for a representative experiment. In brief, HeLa cells transfected with pan-OGT siRNA in high glucose conditions (25 mm glucose) showed enhanced mitochondrial respiration as evidenced by a significant increase in basal OCR (Fig. 3= 12 wells/condition, Bonferroni correction for multiple comparisons. *, < 0.05; PECAM1 **, < 0.01; ***, < 0.001 NT siRNA. However, our image-based analysis indicated that cells with reduced mOGT cultivated in high glucose contained significantly less mitochondria than control cells (Fig. 2and 3= 12 wells/condition, one-way ANOVA, Bonferroni correction for multiple comparisons. *, < 0.05; **, < 0.01; ***, < 0.001 NT siRNA. A significant decrease in mitochondrial content material was also observed when cells cultivated in the absence of glucose (galactose-containing medium), were treated with mOGT siRNA 1, mOGT siRNA 2, and pan-OGT siRNA as compared with cells treated with NT siRNA. The mean and S.D. of percentages of cytosol occupied by mitochondria in galactose-fed cells (determined after staining with Mitotracker Green FM) were 33.9 2.1% PHA-680632 for NT siRNA, 17.9 2.0% for mOGT siRNA 1, 19.2 2.7% PHA-680632 for mOGT siRNA 2, and 27.0 2.1% for pan-OGT siRNA (both mOGT siRNAs < 0.0001 NT siRNA; pan-OGT siRNA < 0.024 NT siRNA). When OCRs were normalized to mitochondrial content material, we did observe nonsignificant raises in baseline OCRs and in the spare respiratory capacity (reserve capacity) of cells transfected with mOGT siRNAs (Fig. 4, and and 20 pmol/min/g of protein in galactose medium (Fig. 4and ?and55< 0.05; **, < 0.01; ***, < 0.001 NT siRNA). To determine the effect of reduced mOGT levels on glycolysis, siRNA-transfected cells were analyzed with the glycolysis stress test. In cells habituated to high glucose conditions and transfected with mOGT or pan-OGT siRNAs NT siRNA, no significant variations were recognized in non-glycolytic acidification (Fig. 5and = 120C150 cells/condition, one-way ANOVA, Bonferroni correction for multiple comparisons. ****, < 0.0001 NT siRNA. Reduction of.