Figure 9. Pellicle-association of aldolase-1 in extracellular Toxoplasma tachyzoites.

(A) Extracellular parasites were disrupted at 4°C by sonication in 25 mM MOPS pH 7.0 containing 5 mM MgCl₂ and 25 mM, 150 mM, or 300 mM KCl and subsequently fractionated into soluble and insoluble fraction by centrifugation. Equal parasite equivalents of the starting material, supernatant, and pellet fractions were separated by SDS-PAGE and analyzed by immunoblotting with monospecific antisera to Toxoplasma aldolase-1 and the membrane skeleton protein IMC1. (B) Extracellular parasites and intracellular parasites were harvested, homogenized in 25 mM MOPS pH 7.0, 5 mM MgCl₂ and 25 mM KCl as described above. The homogenate was fractionated into soluble and particulate material by centrifugation as above. The particulate fraction was subsequently extracted with 1% TX100 in the homogenization buffer and separated by centrifugation into detergent-soluble and insoluble fraction as described above. Equal parasite equivalents were analyzed by SDS-PAGe and immunoblotting with antisera to Toxoplasma aldolase-1, the membrane skeleton protein IMC1, the cytoplasmic protein CDPK1 and the plasma membrane protein SAG1. The starting material (TOT), supernatant (SN), and pellet (P) fractions were separated by SDS-PAGE and immuno-blotted with antisera to the indicated proteins.