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. 2018 Oct 2;9(5):e01510-18. doi: 10.1128/mBio.01510-18

FIG 2.

FIG 2

Generation and validation of a TgFER2 conditional knockdown (cKD) parasite. (A) Schematic representation of single homologous promoter replacement with the anhydrous tetracycline (ATc)-regulatable promoter TetO7sag4. Note that a Myc epitope tag is simultaneously added on the N terminus. Sites of diagnostic primer pairs used in panel B are indicated. (B) Diagnostic PCR of the parent line (TaTiΔKu80) and the FER2-cKD promoter replacement line using the primer pairs depicted in panel A. (C) Western blot demonstrating the conditional expression of the Myc-tagged TgFER2 allele. TgFER2 is downregulated to undetectable levels after 48 h of ATc treatment. Anti-IMC1 is used as a loading control. (D) Immunofluorescence demonstrating the loss of Myc-TgFER2 expression upon ATc treatment for 20 h. Parasites were fixed with 100% methanol. DAPI (4′,6-diamidino-2-phenylindole) labels DNA, and anti-SAG1 marks the plasma membrane. (E) Plaque assays of the parent (TaTiΔKu80) and FER2-cKD lines ± ATc treatment for the times indicated. No plaques are observed upon loss of TgFER2 expression.