FIG. 6.

(A) AT and aerolysin bind to SAG1. Triton X-114 detergent-phase fractions from RH, P(LK), and P(LK)sag1 parasites were analyzed by SDS-PAGE followed by silver staining, AT overlay, aerolysin overlay, and immunoblotting with MAbs against SAG1 and SAG3. The SAG1 and SAG3 immunoblots were performed sequentially, and the data were combined digitally into the single panel shown. A 32-kDa toxin-binding band is observed in RH and P(LK) extracts that comigrates precisely with SAG1 on the corresponding immunoblot. Relatively more aerolysin binds to this protein [in both RH and P(LK) extracts] than does AT. SAG1-deficient parasites lack both immunoreactive SAG1 (as expected) and the 32-kDa toxin-binding protein. No other differences in the toxin-binding profiles were observed between P(LK) and the SAG1-deficient P(LK) parasites. Note that an additional 33-kDa AT-binding protein (asterisk) was observed in P(LK)sag1 parasites that was not observed in RH parasites. The P(LK) and P(LK)sag1 parasites also contained two aerolysin-binding bands not detected in RH parasites (arrowheads). Within each panel, equal amounts of protein were loaded per lane. Numbers on the left indicate molecular mass in kilodaltons. (B) AT and aerolysin bind to SAG3. Triton X-114 detergent-phase extracts from RH(ST) and SAG3-deficient RH(ST) parasites were analyzed as described above. A 38-kDa toxin-binding band is seen in RH(ST) extracts that comigrates precisely with SAG3 on the immunoblot. This 38-kDa toxin-binding band is absent in the RH(ST)ΔSAG3 parasites, which, as expected, also lack immunoreactive SAG3. Numbers on the left indicate molecular mass in kilodaltons.