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[Preprint]. 2024 Oct 29:2023.09.29.560185. Originally published 2023 Sep 29. [Version 2] doi: 10.1101/2023.09.29.560185

PMC10557770.1; 2023 Sep 29
PMC10557770.2; 2024 Oct 29
PMC10557770.3; 2024 Nov 8

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Figure 2. — Endogenous epitope tagging and localization of TgLaforin.

A, Schematic depicting the TgLaforin 3xHA-epitope tagging strategy. X = stop codon. B, Successful tagging of TgLaforin (62 kDa) was verified using immunoblot analysis with an α-HA antibody with SAG1 used as a loading control. C, IFA of Wild type (WT) and TgLaforin-3XHA (TgLaf-HA) tagged parasites with α-HA antibody under tachyzoite and in vitro bradyzoite conditions. Stage conversion is confirmed by Dolichos lectin (DBA) staining. D, Western blot analysis of TgLaforin expression levels in tachyzoites (T) and in vitro bradyzoites (B). Expression of TgLaf-HA is absent under in vitro bradyzoite conditions. GAP45 is the loading control. Decrease in SAG1 alongside increase in SRS9 confirms tachyzoite to bradyzoite conversion. E, IFA of TgLaforin-HA colocalization with PAS. Pearson’s coefficient: 0.765. F, IFA of TgLaforin-HA colocalization with IV58B6. Pearson’s coefficient: 0.737. All scale bars = 5 μm.