Figure 4.

Expression of PbCTRP during ookinete maturation. (a) Induction of PbCTRP transcript (arrow) by in vitro culture of the ookinete. Poly A(+) RNA was extracted from parasites that were purified from the same infected blood (parasitemia 10.5%) before (0 h) and after ookinete culture (16 h). Northern blot (0.1 μg per lane) was hybridized with a probe for PbCTRP (left) and rehybridized with a control probe for Pbs 21 (arrowhead, right). (b) PbCTRP production during ookinete maturation. The infected blood was cultured for ookinete maturation and collected at different time intervals as indicated above each lane. Parasites were purified by erythrocyte lysis in 0.83% NH₄Cl and lysed in SDS-PAGE sample buffer. The samples (containing ∼10⁶ ookinetes per lane) were separated by SDS-PAGE (in a 0–20% gradient) under nonreducing conditions and blotted onto the nitrocellulose membrane for immunodetection using anti-PbCTRP rabbit polyclonal antibody and then alkaline phosphatase–conjugated goat anti–rabbit IgG. (c) Metabolic labeling of ookinete proteins and immunoprecipitation of PbCTRP. Parasites were cultured with [³⁵S]methionine/cysteine (from 5–22 h after exflagellation) and homogenized in the extraction buffer. Labeled PbCTRP (arrowhead) was immunoprecipitated from the extracted proteins using anti-PbCTRP antibody. Samples were separated by SDS-PAGE (in a 0–20% gradient) and visualized in the BAS 2000 system. Lane 1, labeled ookinete proteins (from ∼5 × 10⁴ ookinetes). Lane 2, the same protein extract after absorbing PbCTRP with anti-PbCTRP antibody. Lane 3, purified labeled PbCTRP by immunoprecipitation (from ∼3 × 10⁵ ookinetes).