Fig. 2.
tRip localization. (A) Coimmunolocalization assays on P. berghei sporozoites. The major sporozoite surface protein CSP and tRip were probed with anti-CSP antibodies and anti-PftRip214–402, respectively, and the sporozoite nucleus was stained with DAPI. (B) Extraction of tRip with Triton X-100 (TX-100). TX-100 selectively removes the plasma membrane and its associated proteins (CSP and tRip), whereas the inner membrane complex is resistant to this treatment (25). Sporozoite integrity is shown by GFP staining, with CSP staining shown to verify successful Triton X-100 extraction. (Scale bars, 2 µm.) (C) Subcellular localization of tRip in asexual blood-stage parasites of P. berghei. The schizont of P. berghei was doubly labeled with mouse anti-MSP-1 antibody (surface marker, red) and rabbit anti-tRip antibody (green). Nuclei were visualized with DAPI (blue). (Scale bars, 5 μm.) (D) Protease protection assays on blood-stage parasites. Assays were performed with schizonts freed from erythrocytes, treated with trypsin, and probed with anti-PftRip214–402 antibody. GFP was used as a cytosolic control, digested by trypsin only under denaturing conditions (+ Triton). (E) Carbonate vs. Triton extraction of membrane proteins from blood-stage parasites (schizonts). Both tRip and the apical membrane antigen 1 (AMA-1) (41) were found in the membrane pellet after Na2Co3 treatment and were released into the supernatant only on Triton treatment.