FIG 3.
E3 and 7F can be expressed as intrabodies and maintain their inhibitory functions. (A) Cytosolic fractions from cells transfected with GTD-specific VHHs show strong binding to TcdB. Vero cells transfected with VHH-encoding plasmids were lysed, and cytosolic fractions were tested for specific binding to TcdB by ELISA. TcdB-coated 96-well plates (colored lines) were incubated with serial dilutions of cytosolic fractions. TcdA-coated wells and cytosolic fractions from untransfected cells (cells only) (black lines) were used as negative controls. (B) Effects of 7F and E3 intrabodies on TcdB-induced Rac1 glucosylation. Vero cells were transfected with a panel of fusion plasmids containing genes for RFP-fused VHH monomers before treatment with TcdB. Cytosolic fractions of transfected Vero cells were assessed by Western blotting for the presence of VHH intrabodies and for Rac1 glucosylation status. Antibodies specific for VHHs or the nonglucosylated form of Rac1 (clone 102) were used for visualization. Total Rac1 (clone 23A8) levels were determined as a loading control. (C) Densitometry analysis of percentage of nonglucosylated Rac1 relative to total Rac1 from the three experiments shown in panel B. *, P < 0.05; ns, not significant.