Figure 2. Binding and oligomerization of PFO and PFO^R468A.

(A) Binding of PFO^β4β5 and PFO^{R468A•β4β5} to human RBCs (4×10⁶/ml in a final volume of 0.5 ml) was measured by flow cytometry. The disulfide locked β4β5 versions of each protein [25] were used to prevent the lysis of the RBCs during flow cytometry. (B). Oligomerization of PFO and PFO^R468A(both toxins were maintained at 440 nM)on human RBCs (concentrations ranged from 2.5×10⁷/ml to 2.5×10⁸/ml in a final volume of 40 µl) was determined using SDS-agarose gel electrophoresis (SDS-AGE) and the proteins were detected with anti-PFO antibody after transfer to nitrocellulose paper. The analyses are representative of at least 3 experiments.