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. 2020 Feb 19;5(1):e00877-19. doi: 10.1128/mSphere.00877-19

FIG 5.

FIG 5

The phenotype of GRA44 knockdown can be complemented. To establish a direct connection between the lack of GRA44 and the phenotype observed, we established a complemented strain by adding an exogenous copy of wild-type GRA44 to the TATi-GRA44(HA) conditional knockdown strain. (A) Diagram of the strategy used to establish a complemented strain, TATi-GRA44(HA)comp. The wild-type copy of GRA44 added to the TATi-GRA44(HA) strain contains a C-terminal MYC epitope tag and is driven by the Toxoplasma tubulin promoter (tub). (B) Western blot of lysates from the TATi-GRA44(HA)comp parasites grown with and without ATc for 48 h. The blots were probed for HA and MYC. (C) Representative IFA images of TATi-GRA44(HA)comp with and without ATc treatment stained for the HA-tagged regulatable GRA44 and the MYC-tagged constitutive exogenous copy. Bars = 2 μm. (D) (Left) Plaque assays were performed with the knockdown [TATi-GRA44(HA)] and the complemented [TATi-GRA44(HA)comp] strains grown without (−) or with (+) ATc for 5 days. (Right) The average percentage of the cell monolayer cleared from biological and experimental triplicates is shown on a bar graph (n = 3; mean ± SD; ****, P < 0.0001; n.s., not significant [significance was determined by one-way analysis of variance, followed by Tukey’s test]). (E) TATi-GRA44(HA) was complemented with an exogenous copy of GRA44 containing TEXEL with a deletion of amino acids 1348 to 1352 and a C-terminal MYC tag [TATi-GRA44(HA)compΔTXL]. Lysates from TATi-GRA44(HA), wild-type complemented strain TATi-GRA44(HA)comp, and ΔTXL complemented strain TATi-GRA44(HA)compΔTXL were analyzed by Western blotting and probed for HA and MYC. (F) Plaque assays were performed with the TATi-GRA44(HA) and TATi-GRA44(HA)compΔTXL strains. Parasites were grown 5 days without (−) or with (+) ATc. The average percentage of the cell monolayer cleared for biological and experimental triplicates is shown on a bar graph (n = 3; mean ± SD; ****, P < 0.0001; n.s., not significant [significance was determined by one-way analysis of variance, followed by Tukey’s test]).