JCS000729 Supplementary Material

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• Supplemental Figure 1 (Adobe PDF) -

  Fig. S1. Glycerol gradient analysis of OST complexes after siRNA treatment. OST complexes were isolated from HeLa cells after mock treatment (A) or transfecttion with siRNAs against ribophorin I (B), STT3A (C) or STT3B (D) and solubilised in 1.5% digitonin (see Fig. 2 and main text). In addition to the analysis presented in the main document, the fractions were analyzed for the presence of the ribophorin II (RibII) and Dad1 subunits of the OST complex. Knockdowns of STT3A and STT3B caused a reduction in Dad1 levels and a partial redistribution of ribophorin II to lighter fractions of the gradient.

• Supplemental Figure 2 (Adobe PDF) -

  Fig. S2. Effect of OST subunit knockdown on signal sequence cleavage. In order to confirm that the effects of the RNAi depletions we observe upon N-glycosylation do not reflect a pleiotropic defect of ER function, the efficiency of signal-sequence cleavage for the soluble secretory protein preprolactin was analysed. Preprolactin was synthesized as described in the legend to Fig. 3A in order to determine the effect of siRNA-mediated knockdown of OST subunits upon the efficiency of signal sequence cleavage at the ER. The precursor was synthesized in the absence of ER derived membranes (lane 7, -SP-cells) to confirm the identity of the unprocessed product, and the location of the signal sequence cleaved (PPL-ss) and the unprocessed (PPL+ss) forms are shown. Signal sequence cleavage was detected under all of the experimental conditions studied (cf. lanes 1-6), and quantification showed that the ratio of signal sequence processed to signal sequence unprocessed forms was not significantly altered by any of the treatments used. Variations in the total amount of prolactin seen (lanes 1-6, see PPL-ss) most likely reflect differences in the amount of ER-derived membrane present in the different incubations and do not affect the ratio of processing. The figures shown represent an average from three independent experiments including the standard error.