Journal: PLOS ONE

Article Title: mGBP2 engages Galectin-9 for immunity against Toxoplasma gondii

doi: 10.1371/journal.pone.0316209

Figure Lengend Snippet: mGBP2 -/- MEFs were reconstituted with GFP-mGBP2 WT or one of the indicated mGBP2 truncation mutants as well as one of the N-terminal mCherry fusion proteins mCherry-mGBP2 or mCherry-Gal9. 10 6 Cells were stimulated with IFN-γ for 16 h and infected with T . gondii ME49 for 4h, MOI 50. Cells were lysed and lysate supernatants were incubated o/n with either RFP-Trap ® beads (A, B) or with GFP-Trap ® beads (C-F) at 4°C. Pull-down (PD) samples and appropriate cell lysate supernatants were subjected to Western Blotting (WB). Blots were stained with α-GFP (A, D, F) or α-mCherry (B, C, E) antibodies. GTP-binding domain (GM), the middle domain and the C-terminal effector domain (ME) or the C-terminal effector domain only (E) (37).

Article Snippet: For pull-down experiments, lysate supernatants of mGBP2 -/- MEFs reconstituted either with WT GFP-mGBP2 or GFP-fused truncated mutants mutants or just transduced with a GFP expressing vector, as an appropriate control and coexpressing mCherry fused with Gal9 or just the mCherry protein, were incubated o/n with RFP-Trap ® or GFP-Trap ® beads (ChromoTek GmbH, Germany) at 4°C.

Techniques: Infection, Incubation, Western Blot, Staining, Binding Assay

Journal: PLOS ONE

Article Title: mGBP2 engages Galectin-9 for immunity against Toxoplasma gondii

doi: 10.1371/journal.pone.0316209

Figure Lengend Snippet: gondii . Recruitment and colocalization of mGBP2 and Gal9 was analyzed after transduction of a GFP-mGBP2 fusion construct in mGBP2 -/- MEFs and additional transduction of mCherry-Gal9. MEFs were seeded and incubated on glass slides, stimulated with IFN-γ for 16 h and subsequently infected with T . gondii ME49 for 2 h. After fixation, infected cells were treated with an α-RFP V H H nanobody conjugated to eGFPBoosterAtto647N and with an α-GFP V H H nanobody conjugated to eGFPBoosterAtto488 for enhancement of the immunofluorescence of mCherry and GFP, respectively. Glass slides were analyzed by STED microscopy. Bars 2 μm. The graphs in the right panel depict a fluorescence intensity analysis of STED images on the far right with the ImageJ software (Fiji) for Atto488 and Atto647 fluorescence signals along the cross sections of PVMs as indicated. The colocalization thresholds were set to 7500-max for the Cherry and 5000-max for GFP in mCherry-Gal9 and GFP-mGBP2 expressing cells. Three representative examples from three independent experiments are shown.

Article Snippet: For pull-down experiments, lysate supernatants of mGBP2 -/- MEFs reconstituted either with WT GFP-mGBP2 or GFP-fused truncated mutants mutants or just transduced with a GFP expressing vector, as an appropriate control and coexpressing mCherry fused with Gal9 or just the mCherry protein, were incubated o/n with RFP-Trap ® or GFP-Trap ® beads (ChromoTek GmbH, Germany) at 4°C.

Techniques: Transduction, Construct, Incubation, Infection, Immunofluorescence, Microscopy, Fluorescence, Software, Expressing