Tachyzoites of T. gondii derived from strain RH were cultivated and genetically manipulated as described previously [66 ]. HCF101 (TGME49_318590) and Nbp35 (TGME49_280730) coding sequences were amplified from T. gondii complementary DNA (cDNA) and cloned in frame with a triple hemagglutinin (HA) epitope tag at the 3′ end into plasmid pDt7s4HA. The constructs were transiently transfected into the T. gondii Δku80/TATi strain [67 (link)] using a BTX ECM 630 electroporator (Harward Apparatus). Confluent human foreskin fibroblasts (HFF) were infected with transfected parasites and fixed after 24 h of infection with 4% formaldehyde and permeabilized with 0.2% Triton X-100. Immunofluorescence microscopy was performed using the primary antibodies anti-HA (Roche), mouse anti- T. gondii mitochondrial F1-adenosinetriphosphatase (ATPase) [68 (link)], and rabbit anti-apicoplast Cpn60 [69 (link)]. Secondary antibodies used were goat anti-rat Alexa Fluor 488, goat anti-mouse Alexa Fluor 546, and goat anti-rabbit Alexa Fluor 546. Images were obtained on an Applied Precision Delta Vision microscope and were deconvolved and adjusted using Softworx software (GE Healthcare).
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