Figure 1.

Construction of TgPDH-E1α deletion and complementation strains. A, schematic showing generation of the Δpdh-e1α and Δpdh-e1α/PDH-E1α (CompPDH-E1α) strains via CRISPR-Cas9–assisted gene engineering. PCR1–PCR5 indicate screening of clonal mutants. B, diagnostic PCRs confirming the Δpdh-e1α and CompPDH-E1α strains. C, immunofluorescent assay showing the presence or absence of PDH-E1α. Samples were stained with anti-TgPDH-E1α (which was generated from mice using a recombinant antigen corresponding to residues 158–304 of TgPDH-E1α) and anti-TgCPN60 antibodies. Scale bars = 1 μm. D, immunoblot checking the expression of TgPDH-E1α (green); TgALD (red) was included as a loading control. The two bands for PDH-E1α in the complement strain correspond to the full-length (∼70 kDa) and mature (∼45 kDa, after removal of the apicoplast targeting sequence) forms of the protein.