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. 2017 May 11;18(13):1305–1316. doi: 10.1002/cbic.201700083

Figure 5.

Figure 5

Increased levels of Neu5Ac affect neither related metabolite levels nor global O‐GlcNAcylation. A) Sialic acid anabolism and catabolism in mammals. Enzymes are italicised: CMP‐Sia synthetase (CMAS), lactate dehydrogenase (LDH). Quantification of B) intracellular and C) cell‐culture lactate levels from WT and CMAS‐deficient mESC and HEK metabolite extracts by HPLC‐MS/MS (nmol per mg protein). D–I) Quantification of intracellular TCA cycle metabolite levels from WT and CMAS‐depleted mESC and HEK metabolite extracts by HPLC‐MS/MS (pmol per mg protein). J) Analysis of global O‐GlcNAcylation. Whole‐cell lysates of WT and CMAS‐deficient mESC and HEK cells were analysed by SDS‐PAGE and western blotting with mAb CTD110.6. Specificity of antibody staining was controlled by preincubation of the primary antibody with N‐Acetylglucosamine (GlcNAc). Anti‐actin staining was used as loading control. All mESC lines were cultured feeder‐free with LIF supplementation to maintain the pluripotent state (Cmas +/+ mESC n=2, Cmas −/− mESC n=3, for all HEK cell lines n=3).