(A) Immunoblot showing that pretreatment of mature schizont-infected cells with pronase E, a broad specificity protease, reduces CLAG3 in the membrane fraction (membr) without affecting freeze–thaw released or Na2CO3-extractable (CO3=) pools. (B) Mean ± S.E.M. fractional reduction of indicated CLAG3 pools upon pronase E treatment, determined from changes in band intensities from matched immunoblots as in (A). *p = 0.01, n = 3. (C) Immunoblots showing membrane fractionation of CLAG3 and Band3, a host membrane marker, in purified merozoites and their schizont-infected progenitor cells. Representative of two independent trials. (D) Similar fractionation studies at indicated stages throughout the P. falciparum bloodstream cycle. While schizont- and trophozoite-infected cells were enriched by the percoll–sorbitol method, ring-infected cells cannot be similarly enriched, presumably accounting for CLAG3 detection in the soluble lane and non-additive fractionation in rings. (E) Mean ± S.E.M. band intensities from three independent trials as in (D). *p < 0.005. (F) Schematic shows PTEX-mediated protein translocation and refolding in host erythrocyte cytosol. Middle, Anti-CLAG3 immunoblots from 13F10 cellular fractions with and without trimethoprim (TMP) (top and bottom blots, respectively). Bar graph shows mean ± S.E.M. fraction of integral membrane CLAG3 (Fint), determined from band intensities. *p < 0.015; n = 3. (G) CLAG3-tv2 fractionation studies using enriched mature infected cells. Top, Anti-HA blot showing that soluble CLAG3 (freeze–thaw and CO3 = lanes) is not susceptible to extracellular protease, but the integral pool (membr) is. The ~40 kDa cleavage product remains membrane embedded. EXP2, an intracellular parasite membrane protein, is primarily integral and is protease insensitive. Aldolase, a parasite cytosolic protein, is quantitatively released by freeze–thaw and carbonate treatment. Representative of more than three trials.