Figure 3.

PA41 inhibits TcdB-induced Rac1 glucosylation in Caco-2 cells but not in vitro. A, in vitro glucosylation of GST-Rac1 (2 μm) by TcdB-027-GTD (10 nm) in the presence of [¹⁴C]UDP-glucose (24 μm) and increasing concentrations of PA41 mAb. Controls are shown in lane 1 (GTD with Rac1) and lane 2 (Rac1 only). B, Rac1 glucosylation by TcdB(WT) or TcdB(Y323H) in the presence of isotype control or PA41 mAb was assessed in Caco-2 cells. Cells that did not receive toxin or antibody were used as controls. C, experiments in B were quantified by densitometry, and the extent of Rac1 glucosylation was determined by normalizing the unglucosylated and total Rac1 levels. Means ± S.D. (n = 3) are shown. D and E, similar experiment performed with Fab fragments. The antibody concentration was doubled to keep the available number of antigen-binding sites similar between the mAb and Fab experiments. Error bars represent S.D.