<p>A. Equal amounts of radioactivity associated with material that was eluted from AG-1 columns with 3M FA as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011675#pone-0011675-g002" target="_blank">Fig. 2A</a> were subjected to QAE-Sephadex chromatography as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011675#s2" target="_blank">Material and Methods</a>. The column was eluted with increasing concentrations of NaCl (indicated on the right hand y axis). Fractions were collected and assayed for radioactivity by scintillation counting. B. Aliquots of the negatively charged oligosaccharide-like material derived from EBV CDG Ig cells were analysed by thin layer chromatography (TLC) before and after treatment with either alkaline phosphatase (<i>P'ase</i>) or endo-β-N-acetylglucosaminidase H (<i>EndoH</i>). Abbreviations: lines to the left of the TLC fluorograms indicate the migration position of the oligosaccharide (Man<sub>7</sub>GlcNAc<sub>2</sub>; M<sub>7</sub>GN<sub>2</sub>) that was derived by mild acid hydrolysis of Man<sub>7</sub>GlcNAc<sub>2</sub>-PP-dolichol isolated from CDG Ig cells. This oligosaccharide was also treated with endoH to yield Man<sub>7</sub>GlcNAc (M<sub>7</sub>GN). The structure of the oligosaccharide moiety known to occur in the Man<sub>7</sub>GlcNAc<sub>2</sub>-PP-dolichol that accumulates in cells from CDG Ig is shown to the right of the TLC (mannose; green circles, N-acetylglucosamine; blue squares). The di-N-acetylchitobiose moiety of this oligosaccharide is sensitive to endoH. C. Aliquots of the negatively charged oligosaccharide-like material derived from DPM synthase-deficient Thy<sup>-1</sup> mouse lymphoma cells were analysed by thin layer chromatography (TLC) before and after treatment with either alkaline phosphatase (<i>P'ase</i>) or 20 mM HCl. The structure of the oligosaccharide moiety known to occur in the Man<sub>5</sub>GlcNAc<sub>2</sub>-PP-dolichol that accumulates in these cells is shown to the right of the TLC (mannose; green circles, N-acetylglucosamine; blue squares). The di-N-acetylchitobiose moiety of this oligosaccharide is not sensitive to endoH. The line to the left of the fluorograph indicates the migration position of Man<sub>5</sub>GlcNAc<sub>2</sub> (M<sub>5</sub>GN<sub>2</sub>) that was released by mild acid acid treatment of Man<sub>5</sub>GlcNAc<sub>2</sub>-PP-dolichol derived from Thy<sup>-1</sup> cells. D. [<sup>14</sup>C]glucose-1-phosphate (Glc1P) and [<sup>14</sup>C]glucose-6-phosphate (Glc6P) were subjected to ion-exchange chromatography on AG-1(acetate) before and after either alkaline phosphatase or mild acid treatment as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011675#s2" target="_blank">Materials and Methods</a>. Neutralised material was assayed by scintillation counting and expressed as a percentage of input radioactivity.</p
