Expression of Pf332 is abolished in 3D7ΔPf332 KO clones or expressed as a smaller, truncated protein in 3D7ΔPf332a and 3D7ΔPf332b clones. (A) Western blot analysis of Pf332 expression in 3D7 parasites, 3D7ΔPf332 KO clones (11G7, 12F3, 13E2, and 13F7), and the 3D7ΔPf332a (3F3 and 12H9) and 3D7ΔPf332b (5G1 and 13F5) clones. Membranes were probed with antibodies raised against the NH₂-terminal region of Pf332. Bands corresponding to full-length Pf332 (Pf332FL), or the size-truncated proteins ΔPf332b and ΔPf332a are indicated. An identical blot was probed for HSP70 as a parasite loading control. Densitometric quantitation with NIH ImageJ open source software (National Institutes of Health, Bethesda, MD) of total Pf332 reactivity for 3D7 and each of the truncation mutants (corrected for equal loading of iRBCs) revealed that all mutants express similar levels of protein, which was approximately half the amount of full-length Pf332 expressed by 3D7 parasites (total Pf332 expressed as percentage of 3D7 = 41%, 39%, 53%, and 44% for clones 3F3, 12H9, 5G1, and 13F5, respectively). (B) Immunofluorescence analysis of iRBCs infected with 3D7, 3D7ΔPf332 (11G7), or 3D7ΔPf332a (3F3). iRBCs were incubated with antibodies raised against the N-terminal (E1) or C-terminal (F19) regions of Pf332, or with the MC marker SBP1. Note that no fluorescence of the MCs or iRBC membrane could be detected in the 3D7ΔPf332 KO clones. Parasite nuclei were counterstained with DAPI.