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[Preprint]. 2023 Dec 14:2023.12.13.571530. [Version 1] doi: 10.1101/2023.12.13.571530

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Fig. 2 — A. HFFs were infected with parasites expressing GRA35 endogenously tagged at the C-terminus with the HA epitope for 16 h. After fixation, the cells were permeabilized with 0.001% digitonin followed by staining with antibodies against SAG1 and the HA epitope. The images are representative of results from 2 independent experiments (scale bar = 5 μm). Arrows indicate a fully permeabilized vacuole and arrowheads denote the vacuole that is not permeabilized.

B. Lysates from Lewis rat BMDMs infected with parasites expressing C-terminal HA-tagged GRA35 or other PVM-localized proteins (GRA23 or GRA15, served as negative controls) were immunoprecipitated using HA antibodies followed by mass spectrometry analysis. The total unique spectrum count of host E3 ubiquitin ligase ITCH is indicated.

C. Lysates of HEK293T cells transiently expressing FLAG-rITCH with the indicated GRA fused with HA epitope were immunoprecipitated using HA antibodies followed by immunoblotting (IB) analysis with the indicated antibodies. 5% of the total lysate was loaded and used as input. The images are representative of results from 2 independent experiments.

D. HEK293T cells transiently expressing FLAG-rITCH and either full-length (FL) or the C-terminus (Ct) of GRA35 fused with the HA epitope were analyzed as in (c). The images are representative of results from 2 independent experiments.

E. HEK293T cells transiently expressing FLAG-rITCH and an HA-tagged GRA35 C-terminal fragment were immunoprecipitated using FLAG antibodies and analyzed as in (c). The images are representative of results from 2 independent experiments.

F. Schematic illustration of the constructs used for the generation of full-length or truncated rat ITCH.

G. HEK293T cells transiently expressing a C-terminal fragment of GRA35 fused with the HA epitope and FLAG-tagged rat ITCH truncations (indicated in f) were immunoprecipitated using HA antibodies and analyzed as in (c). The images are representative of results from 2 independent experiments.

H. Lewis rat BMDMs infected with GFP-expressing Toxoplasma for 4h were stained with antibodies against ITCH. The images are representative of results from wild-type parasite infected BMDMs (scale bar = 10 μm). The percentage of vacuoles coated with ITCH was quantified as shown on the right. Data are displayed as mean ± SD with independent experiments (n = 3) indicated by the same color dots. Significance was determined with one-way ANOVA with Tukey’s multiple comparisons test. Arrowhead indicates the vacuole coated with ITCH.