Figure 6

Clone c1 competes with AP65-HA for binding. Part A (panel A1) is an immunoblot of a ligand assay of MS-74 VECs pretreated with c1 recombinant protein for 30 min at RT followed by the addition of trichomonad lysate containing AP65-HA and incubated for 1 h. After SDS-PAGE using 8% acrylamide gels and immunoblotting, membranes were probed with anti-HA mAb to detect bound AP65-HA. Purified rabbit anti-AP65 polyclonal IgG (Figure 4) was used to detect c1. Lane 1 is control detergent lysate of T016 isolate expressing AP65-HA showing wild type AP65 (lower band) and the episomally-expressed fusion AP65-HA (upper band). Lane 2 is control VECs without pretreatment with c1. Lanes 3 and 4 are VECs pretreated with 1 μg and 10 μg of c1, respectively. There is visible inhibition of binding of AP65-HA by pretreatment with 10 μg c1. Panel A2 is an immunoblot to detect clone c1 from the same experiment of A1 to show the binding of the recombinant clone c1 to VECs. Part B is a densitometric scan of the AP65-HA immunoblot bands from part A1 to visually present quantitatively the relative percentage of bound AP65-HA. Error bars indicate standard deviations. Competition with 10 μg was significant (p < 0.05). Panel C1 is an immunoblot of a ligand assay of pretreated T. vaginalis with c1 recombinant protein and handled identically as in part A. Trichomonads were incubated with 1 μg (lane 2) and 10 μg (lane 3) clone c1 before adding extract with AP65-HA. Lane 1 is untreated, control trichomonads without c1 addition. After SDS-PAGE and immunoblotting, AP65-HA was detected with mAb to HA. Panel C2 detects clone c1 bound to parasites as in A2.