Fig. 4. Endosome-to-Golgi transport of Shiga toxin in HeLa cells. HeLa cells were grown with (control) and without (dyn^K44A) tetracycline for 48 hours and with and without 2 mM butyric acid (ba) for the last 24 hours. (A) Sulfation of STxB-Sulf₂ was analysed by incubating the cells with radioactive sulfate for 3 hours at 37°C before STxB-Sulf₂ (2.8 µg ml^–1) was added for 15 minutes or 1 hour. The cells were subsequently washed, lysed and immunoprecipitated with rabbit anti-Shiga toxin antibodies. The adsorbed material was analysed by 12% SDS-PAGE before autoradiography. Quantified average signal intensities (as percentage of the value in control cells) from two independent experiments are shown (A, bottom). As shown, butyric acid strongly increased the sulfation and expression of mutant dynamin gave a strong reduction. (B) In parallel, the internalization of Shiga toxin for the different conditions in (A) was analysed by incubating the cells with TAG- and biotin-labelled Shiga toxin (5 ng ml^–1) for 15 minutes or 1 hour. The SS-linked biotin on the cell-surface-bound toxin was then removed by incubating the cells with 0.1 M MESNa for 1 hour at 0°C. Subsequently, the cells were washed and lysed, and the amounts of TAG- and biotin-labelled Shiga toxin in the lysates were measured using streptavidin beads and an Origen Analyzer. Average values from these two experiments are shown as the percentage of the value in control cells. As shown, butyric-acid treatment almost doubled the amount of endocytosed toxin. (C) The effect of expressing mutant dynamin on the endosome-to-Golgi transport of Shiga toxin (shown as percentage of the value in control cells) was calculated by correcting the average signal intensities in (A) for the amount of Shiga toxin that was internalized in (B). The error bars show the standard error of the mean from the two experiments.