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. 2014 Apr 16;289(21):14796–14811. doi: 10.1074/jbc.M114.554378

FIGURE 6.

FIGURE 6.

Glycolysis pathway is blocked in Nmnat3gt/gt erythrocytes. A, metabolites in the glycolysis pathway and pentose phosphate pathway were measured using RBC samples obtained from 8-week-old WT and Nmnat3gt/gt mice (n = 4 for each group). Glc-6-P/Fru-6-P (glucose 6-phospate/fructose 6-phosphate), fructose 1,6-bisphosphate (Fru-1,6-BP), dihydroxyacetone phosphate (DHAP), GAP, 2,3-bisphosphoglycerate (2,3-BPG), 3-phosphoglycerate (3PG), phosphoenolpyruvate (PEP), pyruvate (PYR), lactate (LAC), 6-phosphogluconate (6PG), PRPP, ribose 5-phosphate/ribulose 5-phosphate (R5P/Ru5P), xylulose 5-phosphate (Xu5P), and sedoheptulose 7-phosphate (S7P) were analyzed by a MRM-based LC-MS/MS method. Data are presented as mean ± S.D. B, enzymatic activities of glycolysis enzymes were assayed with RBC lysate prepared from 8-week-old WT and Nmnat3gt/gt mice (n = 4 for each group). Phosphofructokinase (PFK), aldolase (ALD), pyruvate kinase (PK), hexokinase (HK), glucose-6-phosphate isomerase (GPI), triose-phosphate isomerase (TPI), phosphoglycerate kinase (PGK), enolase (ENOL), and lactate dehydrogenase (LDH) were measured by the methods recommended by International Committee for Standardization in Hematology. C, immunoblot analysis of phosphofructokinase and GAPDH in mature erythrocytes from WT and Nmnat3gt/gt mice. β-Actin was used as loading control. Proteins were extracted from three mice for each group. D, enzymatic activities of pentose phosphate pathway enzymes (Glc-6-PD, glucose-6-phosphate dehydrogenase; 6PGD, 6-phosphogluconolactone dehydrogenase) were assayed with RBC lysate prepared from 8-week-old WT and Nmnat3gt/gt mice (n = 4 for each group). E, enzymatic activities of adenylate kinase (AK) and adenosine deaminase (ADA) were assayed with RBC lysate prepared from 8-week-old WT and Nmnat3gt/gt mice (n = 4 for each group). F, measurement of acetyl-CoA level in RBC prepared from WT and Nmnat3gt/gt mice (n = 4 for each group). G, acetylation status of erythrocytes protein. RBC lysates were separated by SDS-PAGE and subjected to immunoblotting (IB) with acetyl-lysine-specific antibodies (Abcam and Cell Signaling). Proteins were extracted from three different mice for each group.