Figure 2. Characterization of Toxoplasma AP endonucleases.

A. Western analysis with affinity-purified anti-TgAPE or anti-TgAPN, each used at a 1:10,000 dilution. The designated amount of Toxoplasma protein lysate is shown. Recombinant (r) protein expressed and purified from E. coli was run as control, and suggests anti-TgAPE binds its protein with greater affinity than anti-TgAPN, yet TgAPN is much more readily detected in lysates derived from tachyzoites. B. IFA of intracellular parasites probed with 1:1,000 dilution of anti-TgAPE (top panels, green) or 1:10,000 dilution anti-TgAPN (lower panels, green). 4′,6-diamidino-2-phenylindole (DAPI) was used as a co-stain to highlight the nuclear compartment (blue). hN= host cell nucleus. Arrowheads point to parasite apicoplast organelle. C. Total AP endonuclease activity in wild-type Toxoplasma lysate, performed in presence of 1.0 mM MgCl₂. Lane 1, 10 nM recombinant human APE1 (PC, positive control); lane 2, DNA alone (NC, negative control); lanes 3–8, increasing amounts of Toxoplasma lysate (100, 250, 500, 1000, 1500 and 3000 ng, respectively). The columns on the graphs below each gel correspond to the percent activity of the sample directly above and are representative of the average percent activity of two independent assays. D. APN activity levels in parasite lysate, as determined by degree of cleaved oligonucleotide. AP endonuclease assays were performed in the presence of 10 mM EDTA to chelate Mg²⁺. Lane 1, 10 nM recombinant TgAPN (PC, positive control); lane 2, DNA alone (NC, negative control); lanes 3–8, increasing amounts of Toxoplasma lysate (100, 250, 500, 1000, 1500 and 3000 ng, respectively).