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Fig. 6 | Orphanet Journal of Rare Diseases

Fig. 6

From: A case of fatal Type I congenital disorders of glycosylation (CDG I) associated with low dehydrodolichol diphosphate synthase (DHDDS) activity

Fig. 6

Down-regulation of DHDDS expression in HepG2 cells. HepG2 cells were transfected with three siRNA targeting DHDDS or a control non-targetting siRNA (nt) and were harvested 4 days later. a Cellular mRNA was extracted and DHDDS cDNA was quantitated by qRT-PCR. b Total adherent cell protein was assayed. c Cells were radiolabeled with [14C]leucine for 30 min, and radioactivity recorded in precipitated cellular proteins was calculated per mg protein and these values were expressed as a percentage of that found in cells transfected with the control non-targetting siRNA. d DHDDS activity was measured in the three cell populations. e HepG2 cells were mock transfected (None), or transfected with either the control non-targetting siRNA (nt) or siRNA(1) targeting DHDDS, and at 2, 4 and 6 days post transfection the cells were metabolically radiolabeled with [2-3H]mannose. Radioactivity associated with DLO (upper panel) and glycoproteins (NLO: middle panel) was measured as described in the legend to Fig. 2c, and the ratio of incorporation of radioactivity into NLO to that into DLO is shown in the lower panel. f Oligosaccharides released from DLO were resolved by TLC and visualized by fluorography. Regions corresponding to the migration positions of the indicated oligosaccharides were eluted from the plate and assayed by scintillation counting. The abbreviations are as follows: G3-1; Glc3Man9GlcNAc2, M9-2; Man9-2GlcNAc2

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