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. 2020 Oct 16;11:5258. doi: 10.1038/s41467-020-18991-8

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a, b PBS or NP-40-treated pellet (P) and supernatant (S) fraction of extracellular parasite were detected with antibodies against GRA2 (a) or GRA7 (b). SAG1 was used as the parasite loading control. The image is representative of 4 independent experiments. c–f HFFs infected with indicated parasites for 4 h (e) or 24 h (c, d, f) were stained with antibodies against GRA5 (c), GRA7 (d), or MAF1 (e, f). The images are representative of results from three independent experiments (scale bar = 5 μm). The percentage of vacuoles with only PV-lumen staining was quantified as shown on the right. g HFFs infected with indicated parasites transiently expressing GRA23-HA were stained with antibodies against SAG1 (green) and the HA epitope (red). The images are representative of results from three independent experiments for WT and Δgra45 parasites with two experiments for Δmyr1 parasites (scale bar = 5 μm). The percentage of vacuoles with only PV-lumen GRA23 staining was quantified as shown on the right. h HFFs infected with indicated parasites transiently expressing GRA44-Myc were stained with antibodies against the Myc epitope. The images are representative of results from three independent experiments (scale bar = 2 μm). The percentage of vacuoles with only cytosolic GRA44 staining was quantified as shown on the right. i, j HFFs infected with indicated parasites transiently expressing GRA16-HA-FLAG (i) or GRA24-HA-FLAG (j) were subjected to the immunofluorescent assay. The images are representative of results from three independent experiments (scale bar = 10 μm). The nuclear intensity of GRA16 (i) or GRA24 (j) was quantified as shown on the right. Data are displayed as mean ± SEM with independent experiments indicated by the same color dots. Statistical analysis was done by one-way ANOVA with Tukey’s multiple comparisons test (c–f, h–j) and two-tailed paired t-test (g) due to different n between Δmyr1 and Δgra45.