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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. 3 — The Irgb2-b1_CIM interface for T. gondii ROP5B binding is polymorphic. a Pull-down of T. gondii ROP5 by GST-Irgb2_CIM but not GST-Irgb1_CIM from RHΔhxgprt tachyzoite detergent lysates (lower left hand panel). The upper left hand panel indicates input of glutathione S-transferase (GST)-fusion proteins in the pull-down. The right hand panels display amounts of ROP5 (upper panel) and GRA7 (lower panel) in the tachyzoite lysate used for the pull-down. b Schematic representation of Irgb2-b1 CIM-BL/6 chimeric variant (Irgb2-b1_Chimera) used in (c, d). c, d Irgb2-b1_CIM interaction with T. gondii ROP5B but not ROP5C is disturbed in case of Irgb2-b1_Chimera. c Protein-fragment complementation assay. Proteins were fused to N-terminal (BlaN) or C-terminal (BlaC) fragments of the reporter protein TEM-1 β-lactamase. Error bars indicate the mean and standard deviation of three independent experiments (right hand panel). One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison was used to test differences between groups; ****p < 0.0001; n.s. not significant. The kinetic of the β-lactamase reaction is shown for one representative experiment (left hand panel). Interaction of Irgb2-b1_CIM with T. gondii ROP5B is almost completely abrogated with Irgb2-b1_Chimera. d 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. Black dotted lines indicate assembly of relevant areas from two different plates. e Intensities of individual vacuoles positive for Irgb2-b1_CIM and Irgb2-b1_Chimera detected by immunofluorescence with anti-Irgb2-b1-specific antiserum in interferon-γ (IFNγ)-induced (200 U ml⁻¹) wild-type (wt) CIM diaphragm-derived cells (DDCs) infected for 2 h with RHΔhxgprt. Intensities of Irgb2-b1_Chimera are significantly reduced at RHΔhxgprt-derived vacuoles. f Frequency of Irgb2-b1_CIM- and Irgb2-b1_Chimera-positive vacuoles detected by immunofluorescence with anti-Irgb2-b1-specific antiserum in IFNγ-induced (200 U ml⁻¹) complemented Irgb2-b1 ko CIM DDCs infected for 2 h with RHΔhxgprt (about 100 vacuoles were counted per experiment). No differences in numbers of Irgb2-b1_CIM- and Irgb2-b1_Chimera-positive vacuoles could be observed. In e and f, error bars indicate the mean and SEM of three independent experiments. Student's t-test was used for two-group comparisons; ****p < 0.0001, n.s. not significant