Fig. 3. Analysis of endocytosis and TGN/Golgi-to-ER transport in Syn5 RNAi cells. (A) Glycosylation analysis. Iodinated STxB-Glyc-KDEL was incubated with mock-transfected or RNAi-transfected HeLa cells for 4 hours. The percentage of glycosylated STxB-Glyc-KDEL was determined from gels, as described (Johannes et al., 1997; Mallard and Johannes, 2003). Glycosylation of STxB-Glyc-KDEL in mock-transfected cells was taken as the maximal possible (100%) value. Mean ± s.e.m. of two to three experiments. (B,C) The endocytosis of (B) STxB and (C) Tf was determined on the same cells. Data represent the percentage of cell-surface inaccessible biotin-tagged STxB or Tf at each time point. Mean ± s.e.m. of four experiments. (D,E) Quantitative analysis of TGN/Golgi-to-ER transport. (D) STxB-Sulf-Glyc-KDEL was internalized into mock-transfected (± tunicamycin) or siRNA-transfected HeLa cells for 2 hours in the presence of radioactive sulfate and then chased for 4 hours at 37°C. Cells were lysed, STxB-Sulf-Glyc-KDEL immunoprecipitated and analyzed by SDS-PAGE. The uppermost band (1) corresponds to the glycosylation product which disappears upon tunicamycin treatment. See text for details. (E) Quantitative analysis by Phosphoimager of the mean of two to three experiments ± s.e.m., as shown in D. Data express the percentages of sulfated STxB-Sulf-Glyc-KDEL that also become glycosylated in each experimental condition.