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. 2008 Feb 13;2(2):e165. doi: 10.1371/journal.pntd.0000165

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Weber et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Crude membrane preparation from tetracycline induced and uninduced trophozoites were labeled with [14]-C UDP-N-acetylglucosamine in a cell free system in the presence of GDP-mannose. GPI biosynthesis intermediates were isolated and analyzed by HPTLC and visualized by fluorography. Panel A, Lane 1-2: GPIs from untreated and tetracycline treated vector control respectively, lane 3-4, GPIs from untreated and tetracycline treated EhPIG-M1 antisense cells line respectively; lane 5 shows the first two intermediates of the GPI-pathway, namely GlcNAc-PI and GlcN-PI, as control. Panel B shows the densitometric analysis of the radiolabeled products as obtained on HPTLC chromatography, I- GlcN-PI and product X for untreated (clear boxes) and tetracycline treated (black boxes) cells carrying the control vector (CTL), or the EhPIG-M1 antisense construct. An increased level of GlcN-PI (the substrate of PIG-M) is observed in extract carrying reduced levels of EhPIG-M1.