Fig. 2
From: Molecular mechanism for the control of virulent Toxoplasma gondii infections in wild-derived mice
Irgb2-b1CIM directly binds T. gondii-derived ROP5B and ROP5C. a Irgb2-b1CIM was co-imunoprecipitated from RHΔhxgprt- but not RHΔrop5-infected CIM diaphragm-derived cells (DDCs) that have been induced with 200 U ml−1 interferon-γ (IFNγ) using a ROP5-specific antibody (upper left hand panel). The lower left hand panel indicates GRA7 association with ROP5 immunoprecipitations. The star indicates a protein unspecifically detected by the antibody used for immunoprecipitation. The middle and right hand panels display amounts of ROP5, GRA7 and Irgb2-b1 in the lysates used for immunoprecipitation. b Pull-down of ROP5 by CIM but not BL/6 GST-Irgb2-b1 from RHΔhxgprt detergent lysates. RHΔrop5 tachyzoite detergent lysates have been included as control (middle left hand panel). The lower panel indicates GRA7 association with ROP5. The upper panel indicates input of glutathione S-transferase (GST)-fusion proteins in the pull-down. The right hand blot shows ROP5 (upper panel) and GRA7 (lower panel) levels in tachyzoite lysates. c Irgb2-b1CIM interacts with ROP5B and ROP5C but not ROP5A 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. The bold black line separates samples from negative controls. d Protein-fragment complementation assay. Proteins were fused to N-terminal (BlaN) or C-terminal (BlaC) fragments of the reporter protein TEM-1 β-lactamase. The increase in absorbance measured at 405 nm indicates restoration of β-lactamase activity after protein/protein interaction. Binding of Irgb2-b1CIM to ROP5B is significantly stronger compared to ROP5C and ROP5A. 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; **p < 0.0025. The kinetic of the β-lactamase reaction is shown for one representative experiment (left hand panel)
