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Fig. 3. Effect of OST subunit knockdown on the N-glycosylation of type I membrane proteins. Transmembrane topology and number of N-linked glycans (branched structures) for the two proteins studied. (A) APP-C99'.1CHO was synthesised as a radiolabelled polypeptide using rabbit reticulocyte lysate supplemented with semi-permeabilised HeLa cells prepared 48 hours after transfection with siRNAs specific for the mRNAs encoding ribophorin I (lane 1), STT3A (lane 2) or STT3B (lane 3), a scrambled form of the ribophorin I siRNA (siScram. RibI) (lane 4), a non-functional control siRNA (siRF) (lane 5) or following mock transfection (lane 7). As a positive control for loss of N-glycosylation, HeLa cells were incubated with 2 µg/ml tunicamycin for 12 hours before isolation on day 2 (lane 6). The resulting glycosylated (+ CHO) and non-glycosylated (– CHO) polypeptides are shown following SDS-PAGE. The relative proportion of glycosylated polypeptide was calculated for each sample and expressed as a percentage of the total protein recovered. The numbers below the lanes are the mean ± s.e.m. of three independent experiments. Levels of N-glycosylation that differ from the mock-treated control by a significance of at least 0.02 are indicated (*). (B) Human glycophorin C was synthesised as in A except that the scrambled ribophorin I siRNA control was omitted. The proportion of N-glycosylated chains was calculated as for A and symbols are as previously described.
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