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. 2017 Feb 1;28(3):411–428. doi: 10.1091/mbc.E16-08-0587

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© 2017 Nagayasu et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 2: — TgDCX gene, protein, and localization in T. gondii. (A) Arrangement of TgDCX (TGGT1_256030; www.toxodb.org) on Toxoplasma strain TGGT1 chromosome VIIb (top), structure of the mRNA (middle), and protein domain organization (bottom). Noncoding regions of the mRNA are indicated by the white boxes. Coding sequence is in yellow. The arrow indicates an alternative start codon. The stop codon is marked by an asterisk. Regions of the protein with significant homology to known eukaryotic proteins are marked by the purple boxes. The arrow indicates the alternative translation initiation site 15 aa residues from the first methionine. Numbers indicate aa residues counting from the first methionine. (B) Western blot with rabbit antibody raised against bacterially expressed recombinant TgDCX. Lane 1 (T.g.), whole-cell lysate of RH parasites, 10 µg of protein, 7 × 10⁶ cells. Lane 2 (HFF), 10 µg of protein from whole-cell lysate of uninfected host cells (HFFs). (C) Images of a transgenic RH line expressing EGFP-β3-tubulin (green) and stained with rabbit anti-TgDCX antibody (red). Top, intracellular parasites fixed and permeabilized with methanol and briefly treated with 10 mM sodium deoxycholate. With this protocol or using nonionic detergent permeabilization, the developing daughter conoids are labeled with anti-TgDCX (yellow arrows), but antibody does not penetrate the adult conoid (magenta arrows). Anti-GFP antibody behaves similarly; in parasites expressing TgDCX-EGFP, daughter conoids can be stained with a GFP antibody, but adult conoids are not labeled (unpublished data), even though the adult conoids are brightly fluorescent from the DCX-EGFP they contain, as in the top left. Bottom, using longer deoxycholate treatment to permeabilize/extract extracellular parasites allows the antibody to penetrate the adult conoid (magenta arrow), but the architecture of the parasite is mostly disrupted. (D) Three EM images of deoxycholate-extracted RH parasites stained with anti-TgDCX antibody and 1.4-nm-gold secondary antibody and silver enhanced. The silver/gold deposits are confined to the conoid. Cortical microtubules are not labeled by the antibody. Middle, the conoid is partially uncoiled, revealing individual conoid fibers with attached gold/silver deposits.