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. 2019 Mar 15;10:1233. doi: 10.1038/s41467-019-09200-2

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© The Author(s) 2019

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. 6 — T. gondii VAND-derived ROP5 escapes targeting by Irgb2-b1_CIM. a p(T108)Irga6_CIM protein intensities are increased at VAND-derived vacuoles. interferon-γ (IFNγ)-induced CIM diaphragm-derived cells (DDCs; 200 U ml⁻¹) were infected for 2 h with indicated T. gondii strains and individual p(T108)Irga6_CIM -positive vacuoles identified with 558 p(T108)Irga6_CIM-specific antiserum. Error bars indicate the mean and SEM of three independent experiments. Kruskal–Wallis test followed by Dunn's multiple comparisons was used to test differences between groups; ****p < 0.0001; n.s. not significant. b Irgb2-b1_CIM intensities are reduced at VAND- and AS28-derived vacuoles. IFNγ-induced CIM DDCs (200 U ml⁻¹) were infected for 2 h with indicated T. gondii strains and individual Irgb2-b1_CIM -positive vacuoles identified with anti-Irgb2-b1-specific antiserum. Error bars indicate the mean and SEM of three independent experiments. Kruskal–Wallis test followed by Dunn's multiple comparisons was used to test differences between groups; ****p < 0.0001; **p < 0.006. c In vitro pull-down with recombinant GST-Irgb2-b1_CIM fusion protein and T. gondii tachyzoite detergent lysates. Pull-down of ROP5 by Irgb2-b1_CIM is reduced with AS28 and completely lost in case of VAND tachyzoite lysates compared to RHΔhxgprt-derived ROP5 (middle left hand panel). VAND GRA7 pull-down is not dependent on ROP5 (lower panel). The upper panel indicates input of GST-Irgb2-b1_CIM in the pull-down. The right hand blot shows ROP5 (upper panel) and GRA7 (lower panel) levels in tachyzoite lysates. All tracks were run on a single gel; vertical white lines indicate excision of irrelevant tracks. d VAND ROP5 isoforms ROP5A, ROP5B1, ROP5B2 and ROP5B3 do not directly interact with Irgb2-b1_CIM or VAND GRA7 in a yeast two-hybrid approach. Proteins were expressed either as fusion to a transcriptional activation domain (AD) from pGAD-C3 or to a DNA-binding domain (BD) from pGBD-C3. Colony growth under 3DO conditions is indicative of protein/protein interaction. Bold black line separates samples from negative controls. e Upper panel, alignment of T. gondii RH and VAND ROP5 amino acid sequences that represent a polymorphic hotspot and have been shown to be involved in binding to Irga6_BL/6. Polymorphic sites are highlighted in blue. Lower panel, phylogenetic analysis and maximum likelihood tree of T. gondii RH and VAND rop5 sequences. Only maximum likelihood bootstrap values above 50 are shown