Figure 7.

Effect of genistein and piceatannol on the sulfation of StxB-Sulf₂. (A) HeLa cells were incubated with radioactive sulfate for 3 h at 37°C, and during the last 30 min, 50 μg/ml genistein or 50 μM piceatannol was added (duplicate samples). Then, StxB-Sulf₂ (1 μg/ml) was added to the medium, and the incubation was continued for 1 h. The cells were subsequently washed, lysed, and immunoprecipitated with rabbit anti-Stx antibody overnight at 4°C. The adsorbed material was analyzed by 12% SDS-PAGE before autoradiography. The experiment shown is representative of at least three independent experiments. Graph, quantification of the amount of sulfated StxB. The error bars show the deviation between the two duplicates. (B) HeLa cells transfected with nonspecific siRNA or Syk siRNA 1 were incubated with radioactive sulfate and then StxB-Sulf₂ as described in A. The Syk mRNA level was reduced by 80–90% after transfection with Syk siRNA 1. Graph, quantification of the amount of sulfated StxB. The error bars show the deviation between two independent experiments.