Journal: Cellular Microbiology

Article Title: Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole

doi: 10.1111/j.1462-5822.2010.01443.x

Figure Lengend Snippet: Time-course of Irga6 and Irgb6 association with T. gondii ME49 PVs. IFNγ-induced C57BL/6 MEFs were infected with T. gondii ME49 strain as described in Experimental procedures . At intervals from 2.5 min to 2 h after infection slides were prepared for staining simultaneously with antibody reagents against Irga6 (mAb 10D7) and Irgb6 (serum A20) using secondary antibodies coupled with the same fluorochrome to enhance the visible signal (A, C) or against Irgb6 (serum A20) alone (B). DAPI was used to stain the nuclei. A. Loading of IRG proteins begins early after cell penetration. Vacuoles with visible accumulations of IRG proteins on the PVM were counted per 1000 host cell nuclei at each time point and presented as a percentage of maximum. The mean of two independent repetitions and the range between them are shown. B. The frequency of Irgb6-positive vacuoles increased with time after infection. In two independent experiments Irgb6-positive PVs were counted out of 10–100 intracellular parasites at different time points after infection. Mean and ranges are given. The 2 min time point was assayed in only one experiment. C. IRG signal intensity at the PVM increased with time after infection. Fluorescent signal intensities of IRG protein (Irgb6 plus Irga6) on individual vacuoles were measured as described in Experimental procedures (see also ) at the times indicated. Neither signal intensities nor heterogeneity were detectably affected by synchronized infection (as described in Experimental procedures ) and thorough removal of free parasites by washing. Open circles: infection was synchronized and free parasites were washed off; closed circles: infection was not synchronized and free parasites were not washed off after inoculation. Twenty-five positive vacuoles were measured at each time point.

Article Snippet: The following immunoreagents were used: rabbit anti-Irga6 antiserum 165/3 ( Martens et al. , 2004 ), mouse anti-Irga6 monoclonal antibodies (mAb) 10E7 and 10D7 ( Papic et al. , 2008 ), mouse anti-Irgm3 monoclonal antibody anti-IGTP (BD Biosciences, 610881), goat anti-Irgb6 antiserum A20 (Santa Cruz Biotechnology, sc-11079), rabbit anti-Irgb6 antiserum 141/1 was raised against recombinant bacterial full-length protein (N. Pawlowski, unpublished data), mouse anti-Irgb6 monoclonal antibody B34 ( Carlow et al. , 1998 ), rabbit anti-Irgm2 antiserum H53/3, raised against the N-terminal peptide of Irgm2 from C57BL/6 and unreactive against Irgm2 from 129 strain mice ( Martens et al. , 2005 and J.C. Howard, unpublished data), rabbit anti-Irgd antiserum 2078/3 ( Martens et al. , 2004 ), rabbit anti-Irgd antiserum 081/1 was raised against bacterially synthesized full-length protein (N. Pawlowski and G. Vopper, unpublished data), rabbit anti-Irgb10 antiserum ( Coers et al. , 2008 ), anti-Irgm1 antiserum L115 B0 ( Martens et al. , 2005 ), anti- T. gondii rabbit (BioGenex, PU125-UPE) and goat antisera (Abcam, ab23507), mouse IgG anti-GRA7 monoclonal antibody ( Bonhomme et al. , 1998 ) and mouse IgM anti-GRA7 monoclonal antibody TxE2 ( Fischer et al. , 1998 ), rabbit anti-ctag1 antiserum 2600 ( Martens et al. , 2005 ), rabbit anti-calnexin antiserum (StressGene, SPA-865), mouse anti-α-tubulin monoclonal antibody (Sigma-Aldrich, T 6074), mouse anti-Ty-tag monoclonal antibody ( Bastin et al. , 1996 , kindly provided by Keith Gull), rat anti-LAMP1 monoclonal antibody (University of Iowa), anti-Akt (Cell Signaling, 9272) and anti-phosphoAkt (Cell Signaling, 9271) rabbit polyclonal antibodies, rabbit anti-PARP1 polyclonal antibody (Cell Signaling, 9542), Alexa 350/488/546/555/647-labelled donkey anti-mouse, rabbit and goat sera (Molecular Probes), donkey anti-rabbit- (GE Healthcare), donkey anti-goat- (Santa Cruz Biotechnology) and goat anti-mouse-HRP (horseradish peroxidase) (Pierce) antisera.

Techniques: Infection, Staining

Journal: Cellular Microbiology

Article Title: Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole

doi: 10.1111/j.1462-5822.2010.01443.x

Figure Lengend Snippet: Influence of duration of IFNγ induction on Irga6 and Irgb6 protein levels and on vacuolar loading. MEFs were induced for different times with IFNγ before infection with T. gondii strain ME49 for 2 h and stained in immunofluorescence against Irga6 and Irgb6. Co-staining against GRA7 was used to determine intracellular parasites. A. and B. The pixel intensities of (A) Irgb6 (serum A20) and (B) Irga6 (serum 165) signals at the PVM of ME49 vacuoles were determined as described in Experimental procedures (see also ) and displayed as a function of IFNγ induction time. Sixty PVs were quantified per time point and the arithmetic means are given as horizontal lines. C. In parallel sample cell lysates from MEFs induced for the indicated times with IFNγ were analysed by Western blot for Irga6 (mAb 10D7) and Irgb6 (mAb B34) expression level relative to calnexin as a loading control.

Article Snippet: The following immunoreagents were used: rabbit anti-Irga6 antiserum 165/3 ( Martens et al. , 2004 ), mouse anti-Irga6 monoclonal antibodies (mAb) 10E7 and 10D7 ( Papic et al. , 2008 ), mouse anti-Irgm3 monoclonal antibody anti-IGTP (BD Biosciences, 610881), goat anti-Irgb6 antiserum A20 (Santa Cruz Biotechnology, sc-11079), rabbit anti-Irgb6 antiserum 141/1 was raised against recombinant bacterial full-length protein (N. Pawlowski, unpublished data), mouse anti-Irgb6 monoclonal antibody B34 ( Carlow et al. , 1998 ), rabbit anti-Irgm2 antiserum H53/3, raised against the N-terminal peptide of Irgm2 from C57BL/6 and unreactive against Irgm2 from 129 strain mice ( Martens et al. , 2005 and J.C. Howard, unpublished data), rabbit anti-Irgd antiserum 2078/3 ( Martens et al. , 2004 ), rabbit anti-Irgd antiserum 081/1 was raised against bacterially synthesized full-length protein (N. Pawlowski and G. Vopper, unpublished data), rabbit anti-Irgb10 antiserum ( Coers et al. , 2008 ), anti-Irgm1 antiserum L115 B0 ( Martens et al. , 2005 ), anti- T. gondii rabbit (BioGenex, PU125-UPE) and goat antisera (Abcam, ab23507), mouse IgG anti-GRA7 monoclonal antibody ( Bonhomme et al. , 1998 ) and mouse IgM anti-GRA7 monoclonal antibody TxE2 ( Fischer et al. , 1998 ), rabbit anti-ctag1 antiserum 2600 ( Martens et al. , 2005 ), rabbit anti-calnexin antiserum (StressGene, SPA-865), mouse anti-α-tubulin monoclonal antibody (Sigma-Aldrich, T 6074), mouse anti-Ty-tag monoclonal antibody ( Bastin et al. , 1996 , kindly provided by Keith Gull), rat anti-LAMP1 monoclonal antibody (University of Iowa), anti-Akt (Cell Signaling, 9272) and anti-phosphoAkt (Cell Signaling, 9271) rabbit polyclonal antibodies, rabbit anti-PARP1 polyclonal antibody (Cell Signaling, 9542), Alexa 350/488/546/555/647-labelled donkey anti-mouse, rabbit and goat sera (Molecular Probes), donkey anti-rabbit- (GE Healthcare), donkey anti-goat- (Santa Cruz Biotechnology) and goat anti-mouse-HRP (horseradish peroxidase) (Pierce) antisera.

Techniques: Infection, Staining, Immunofluorescence, Western Blot, Expressing, Control

Journal: Cellular Microbiology

Article Title: Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole

doi: 10.1111/j.1462-5822.2010.01443.x

Figure Lengend Snippet: Vacuolar loading of IRG proteins is independent of major signalling systems and microtubules. C57BL/6 MEFs were induced with IFNγ and treated as described in Experimental procedures with inhibitors of PI3-kinase (wortmannin and LY294002), G protein-coupled receptors (pertussis toxin), caspases (z-VAD-fmk) and microtubule polymerization (nocodazole). Multiple TLR-mediated signals were excluded in IFNγ-induced MEFs from MyD88-deficient mice. The efficacy of each treatment was assayed as described in Experimental procedures and as shown in . Untreated, treated and MyD88-deficient cells were infected with T. gondii ME49 strain for 2 h and stained separately with antibody reagents against Irga6 (mAb 10D7) and Irgb6 (serum A20). The frequency of vacuoles detectably positive for Irga6 and Irgb6 was calculated as a percentage from 200–400 intracellular parasites. One representative experiment out of two independent repetitions is shown.

Article Snippet: The following immunoreagents were used: rabbit anti-Irga6 antiserum 165/3 ( Martens et al. , 2004 ), mouse anti-Irga6 monoclonal antibodies (mAb) 10E7 and 10D7 ( Papic et al. , 2008 ), mouse anti-Irgm3 monoclonal antibody anti-IGTP (BD Biosciences, 610881), goat anti-Irgb6 antiserum A20 (Santa Cruz Biotechnology, sc-11079), rabbit anti-Irgb6 antiserum 141/1 was raised against recombinant bacterial full-length protein (N. Pawlowski, unpublished data), mouse anti-Irgb6 monoclonal antibody B34 ( Carlow et al. , 1998 ), rabbit anti-Irgm2 antiserum H53/3, raised against the N-terminal peptide of Irgm2 from C57BL/6 and unreactive against Irgm2 from 129 strain mice ( Martens et al. , 2005 and J.C. Howard, unpublished data), rabbit anti-Irgd antiserum 2078/3 ( Martens et al. , 2004 ), rabbit anti-Irgd antiserum 081/1 was raised against bacterially synthesized full-length protein (N. Pawlowski and G. Vopper, unpublished data), rabbit anti-Irgb10 antiserum ( Coers et al. , 2008 ), anti-Irgm1 antiserum L115 B0 ( Martens et al. , 2005 ), anti- T. gondii rabbit (BioGenex, PU125-UPE) and goat antisera (Abcam, ab23507), mouse IgG anti-GRA7 monoclonal antibody ( Bonhomme et al. , 1998 ) and mouse IgM anti-GRA7 monoclonal antibody TxE2 ( Fischer et al. , 1998 ), rabbit anti-ctag1 antiserum 2600 ( Martens et al. , 2005 ), rabbit anti-calnexin antiserum (StressGene, SPA-865), mouse anti-α-tubulin monoclonal antibody (Sigma-Aldrich, T 6074), mouse anti-Ty-tag monoclonal antibody ( Bastin et al. , 1996 , kindly provided by Keith Gull), rat anti-LAMP1 monoclonal antibody (University of Iowa), anti-Akt (Cell Signaling, 9272) and anti-phosphoAkt (Cell Signaling, 9271) rabbit polyclonal antibodies, rabbit anti-PARP1 polyclonal antibody (Cell Signaling, 9542), Alexa 350/488/546/555/647-labelled donkey anti-mouse, rabbit and goat sera (Molecular Probes), donkey anti-rabbit- (GE Healthcare), donkey anti-goat- (Santa Cruz Biotechnology) and goat anti-mouse-HRP (horseradish peroxidase) (Pierce) antisera.

Techniques: Infection, Staining

Journal: Cellular Microbiology

Article Title: Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole

doi: 10.1111/j.1462-5822.2010.01443.x

Figure Lengend Snippet: Atg5 influences loading of IRG proteins onto T. gondii PVs. A. IRG protein association with T. gondii ME49 PVs is reduced in Atg5 −/− fibroblasts. Wt and Atg5 −/− fibroblasts were induced for 24 h with IFNγ and infected with T. gondii ME49 strain for 2 h. Irga6-, Irgb6- and Irgd-positive vacuoles were detected by staining with mAb 10D7, serum A20 and serum 081/1 respectively. A total of 400–700 intracellular parasites were scored for each IRG protein in each cell line in 2–3 independent experiments. For statistical analysis the results for each condition were pooled. For all three IRG proteins the difference between wt and Atg5 −/− was highly significant by chi-squared test, P < 0.001, indicated by *** in the figure). B. The intensity of Irgb6 and Irga6 vacuolar loading is reduced in Atg5 −/− cells. Loading intensity was measured as described in Experimental procedures on at least 40 vacuoles from the experiment shown in (A). Horizontal bars represent the arithmetic mean values. By the Mann–Whitney test, for Irgb6 P < 0.01 for the difference between wt and Atg5 −/− , indicated by ** in the figure, and for Irga6 P < 0.05, indicated by * in the figure). C. Irga6, Irgb6, Irgd and Irgm2 protein levels are reduced in Atg5 −/− MEFs while Irgm1 and Irgm3 are unaffected. Wt and Atg5 −/− fibroblasts were induced with IFNγ for 24 h and analysed by Western blot with antibody reagents detecting the following IRG proteins: Irga6 (mAb 10D7), Irgb6 (mAb B34), Irgd (serum 2078/3), Irgm2 (serum H53/3), Irgm1 (serum L115 BO) and Irgm3 (mAb anti-IGTP). D. IRG proteins form aggregates in IFNγ-induced Atg5 −/− MEFs. Cells were induced with IFNγ, infected with T. gondii ME49 strain and prepared for microscopical analysis as described in (A). Rabbit anti- Toxoplasma serum (upper and middle panels) or anti-GRA7 (lower panel) monoclonal antibody was used to identify the pathogen. Arrows indicate intracellular parasites. The arrowheads indicate the IRG protein aggregates. PhC, phase contrast.

Article Snippet: The following immunoreagents were used: rabbit anti-Irga6 antiserum 165/3 ( Martens et al. , 2004 ), mouse anti-Irga6 monoclonal antibodies (mAb) 10E7 and 10D7 ( Papic et al. , 2008 ), mouse anti-Irgm3 monoclonal antibody anti-IGTP (BD Biosciences, 610881), goat anti-Irgb6 antiserum A20 (Santa Cruz Biotechnology, sc-11079), rabbit anti-Irgb6 antiserum 141/1 was raised against recombinant bacterial full-length protein (N. Pawlowski, unpublished data), mouse anti-Irgb6 monoclonal antibody B34 ( Carlow et al. , 1998 ), rabbit anti-Irgm2 antiserum H53/3, raised against the N-terminal peptide of Irgm2 from C57BL/6 and unreactive against Irgm2 from 129 strain mice ( Martens et al. , 2005 and J.C. Howard, unpublished data), rabbit anti-Irgd antiserum 2078/3 ( Martens et al. , 2004 ), rabbit anti-Irgd antiserum 081/1 was raised against bacterially synthesized full-length protein (N. Pawlowski and G. Vopper, unpublished data), rabbit anti-Irgb10 antiserum ( Coers et al. , 2008 ), anti-Irgm1 antiserum L115 B0 ( Martens et al. , 2005 ), anti- T. gondii rabbit (BioGenex, PU125-UPE) and goat antisera (Abcam, ab23507), mouse IgG anti-GRA7 monoclonal antibody ( Bonhomme et al. , 1998 ) and mouse IgM anti-GRA7 monoclonal antibody TxE2 ( Fischer et al. , 1998 ), rabbit anti-ctag1 antiserum 2600 ( Martens et al. , 2005 ), rabbit anti-calnexin antiserum (StressGene, SPA-865), mouse anti-α-tubulin monoclonal antibody (Sigma-Aldrich, T 6074), mouse anti-Ty-tag monoclonal antibody ( Bastin et al. , 1996 , kindly provided by Keith Gull), rat anti-LAMP1 monoclonal antibody (University of Iowa), anti-Akt (Cell Signaling, 9272) and anti-phosphoAkt (Cell Signaling, 9271) rabbit polyclonal antibodies, rabbit anti-PARP1 polyclonal antibody (Cell Signaling, 9542), Alexa 350/488/546/555/647-labelled donkey anti-mouse, rabbit and goat sera (Molecular Probes), donkey anti-rabbit- (GE Healthcare), donkey anti-goat- (Santa Cruz Biotechnology) and goat anti-mouse-HRP (horseradish peroxidase) (Pierce) antisera.

Techniques: Infection, Staining, MANN-WHITNEY, Western Blot

Journal: Cellular Microbiology

Article Title: Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole

doi: 10.1111/j.1462-5822.2010.01443.x

Figure Lengend Snippet: IRG proteins load in a consistent hierarchy on to the PV of T. gondii ME49 strain. A. Each IRG protein loads onto a characteristic proportion of vacuoles. Quantification of IRG-positive PVs (%) observed in IFNγ-induced 2 h T. gondii ME49 infected MEFs and gs3T3 cells assayed by immunocytochemistry using antibody reagents described in and in Experimental procedures . At least three independent experiments were assayed and pooled and a minimum of 500 PVs counted for each IRG protein (error bars indicate the standard deviation between individual experiments). The statistical significances of the differences recorded were determined by Student's t -test and are shown on the figure (*** P < 0.001; * P < 0.05). B. IRG proteins do not load at random onto each vacuole. IRG proteins loaded onto T. gondii PV were detected by co-staining with pairs of specific antibodies directed against IRG proteins at different positions in the hierarchy, using specific secondary reagents carrying different fluorochromes. Vacuoles loaded with one IRG protein were scored for possession of the second and vice versa. Vacuoles loaded with neither IRG protein were not included in the analysis. At least 100 positive vacuoles were counted for each pair of IRG proteins. Red bar segments give the percentage of vacuoles loaded with both IRG proteins in a given pair, while the green and black bar segments give respectively the percentages loaded with only the lower or only the higher member. The PV loading of pairs of IRG proteins is very strongly correlated such that nearly every vacuole loaded with an IRG protein lower down the hierarchy is also loaded with an IRG protein higher in the hierarchy. The full data are shown in . C. Irgb6 loads more heavily onto T. gondii vacuoles at early time points than Irga6. C57BL/6 MEFs were induced with IFNγ and infected with T. gondii ME49 strain. At indicated times after infection Irgb6 and Irga6 vacuole loading intensities were analysed simultaneously with specific primary antibodies (Irgb6, serum A20; Irga6, mAb 10D7) detected with secondary antibodies labelled with different fluorochromes. D. Irgb6 loads before Irgd on to the T. gondii ME49 strain PV. C57BL/6 MEFs were induced with IFNγ and transfected simultaneously with constructs expressing Irgb6-FLAG-EGFP and Irgd-ctag1-Cherry. After 24 h, cells were infected with T. gondii ME49 strain in microscope slide chambers and observed by live cell imaging for the accumulation of IRG proteins. Successive 1 min frames from one vacuole show Irgb6-FLAG-EGFP visibly loading several minutes before Irgd-ctag1-Cherry. E. Absence of Irga6 does not affect the proportion of vacuoles loaded with Irgb6 or Irgd. Irga6 −/− and wt MEFs were induced with IFNγ and infected with T. gondii strain ME49. 2 h after infection cells were stained with appropriate antibody reagents and the proportion of Irgb6 (mAb B34) and Irgd (serum 081/1) labelled vacuoles (out of 300 for each IRG protein in two independent experiments) was recorded. F. Intensity of PV loading by Irgb6 is significantly reduced in Irga6 −/− relative to wt MEFs ( P < 0.001 by Mann–Whitney test, indicated by *** on the figure). IFNγ-induced Irga6 −/− and wt MEFs were infected with T. gondii ME49 strain. Two hours after infection slides were stained for Irgb6 (B34), Irgd (081/1) and Irgm2 (H53/3). At least 50 vacuoles loaded with each IRG protein were assayed for loading intensity from both cell types. The arithmetic means are given as horizontal lines.

Article Snippet: The following immunoreagents were used: rabbit anti-Irga6 antiserum 165/3 ( Martens et al. , 2004 ), mouse anti-Irga6 monoclonal antibodies (mAb) 10E7 and 10D7 ( Papic et al. , 2008 ), mouse anti-Irgm3 monoclonal antibody anti-IGTP (BD Biosciences, 610881), goat anti-Irgb6 antiserum A20 (Santa Cruz Biotechnology, sc-11079), rabbit anti-Irgb6 antiserum 141/1 was raised against recombinant bacterial full-length protein (N. Pawlowski, unpublished data), mouse anti-Irgb6 monoclonal antibody B34 ( Carlow et al. , 1998 ), rabbit anti-Irgm2 antiserum H53/3, raised against the N-terminal peptide of Irgm2 from C57BL/6 and unreactive against Irgm2 from 129 strain mice ( Martens et al. , 2005 and J.C. Howard, unpublished data), rabbit anti-Irgd antiserum 2078/3 ( Martens et al. , 2004 ), rabbit anti-Irgd antiserum 081/1 was raised against bacterially synthesized full-length protein (N. Pawlowski and G. Vopper, unpublished data), rabbit anti-Irgb10 antiserum ( Coers et al. , 2008 ), anti-Irgm1 antiserum L115 B0 ( Martens et al. , 2005 ), anti- T. gondii rabbit (BioGenex, PU125-UPE) and goat antisera (Abcam, ab23507), mouse IgG anti-GRA7 monoclonal antibody ( Bonhomme et al. , 1998 ) and mouse IgM anti-GRA7 monoclonal antibody TxE2 ( Fischer et al. , 1998 ), rabbit anti-ctag1 antiserum 2600 ( Martens et al. , 2005 ), rabbit anti-calnexin antiserum (StressGene, SPA-865), mouse anti-α-tubulin monoclonal antibody (Sigma-Aldrich, T 6074), mouse anti-Ty-tag monoclonal antibody ( Bastin et al. , 1996 , kindly provided by Keith Gull), rat anti-LAMP1 monoclonal antibody (University of Iowa), anti-Akt (Cell Signaling, 9272) and anti-phosphoAkt (Cell Signaling, 9271) rabbit polyclonal antibodies, rabbit anti-PARP1 polyclonal antibody (Cell Signaling, 9542), Alexa 350/488/546/555/647-labelled donkey anti-mouse, rabbit and goat sera (Molecular Probes), donkey anti-rabbit- (GE Healthcare), donkey anti-goat- (Santa Cruz Biotechnology) and goat anti-mouse-HRP (horseradish peroxidase) (Pierce) antisera.

Techniques: Infection, Immunocytochemistry, Standard Deviation, Staining, Transfection, Construct, Expressing, Microscopy, Live Cell Imaging, MANN-WHITNEY

Journal: Cellular Microbiology

Article Title: Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole

doi: 10.1111/j.1462-5822.2010.01443.x

Figure Lengend Snippet: Loading of Irga6 at the T. gondii ME49 strain PV is enhanced by the presence of other IRG proteins of the GKS group. gs3T3-Irga6 cells were induced with IFNγ or Mifepristone. At the same time, Mifepristone-induced cells were transfected with pools of constructs (see Experimental procedures for experimental details) expressing either the three GMS proteins, (Irgm1, Irgm2 and Irgm3) alone to permit access of Irga6 to the PV (3GMS) or, in addition to the 3GMS proteins, also Irgb6 (3GMS + b6), Irgd (3GMS + d) or both Irgb6 and Irgd (5IRGs). After 24 h, cells were infected with T. gondii ME49 strain for 2 h. Irga6 was detected at the PV in transfected cells using mAb 10E7 in immunofluorescence. Transfected cells were identified by staining for Irgm2 with the H53/3 serum. The arithmetic means are given as horizontal lines. Vacuolar loading of Irga6 was significantly enhanced by addition of Irgb6 or Irgd. The P -values are given on the figure (*** P < 0.001).

Article Snippet: The following immunoreagents were used: rabbit anti-Irga6 antiserum 165/3 ( Martens et al. , 2004 ), mouse anti-Irga6 monoclonal antibodies (mAb) 10E7 and 10D7 ( Papic et al. , 2008 ), mouse anti-Irgm3 monoclonal antibody anti-IGTP (BD Biosciences, 610881), goat anti-Irgb6 antiserum A20 (Santa Cruz Biotechnology, sc-11079), rabbit anti-Irgb6 antiserum 141/1 was raised against recombinant bacterial full-length protein (N. Pawlowski, unpublished data), mouse anti-Irgb6 monoclonal antibody B34 ( Carlow et al. , 1998 ), rabbit anti-Irgm2 antiserum H53/3, raised against the N-terminal peptide of Irgm2 from C57BL/6 and unreactive against Irgm2 from 129 strain mice ( Martens et al. , 2005 and J.C. Howard, unpublished data), rabbit anti-Irgd antiserum 2078/3 ( Martens et al. , 2004 ), rabbit anti-Irgd antiserum 081/1 was raised against bacterially synthesized full-length protein (N. Pawlowski and G. Vopper, unpublished data), rabbit anti-Irgb10 antiserum ( Coers et al. , 2008 ), anti-Irgm1 antiserum L115 B0 ( Martens et al. , 2005 ), anti- T. gondii rabbit (BioGenex, PU125-UPE) and goat antisera (Abcam, ab23507), mouse IgG anti-GRA7 monoclonal antibody ( Bonhomme et al. , 1998 ) and mouse IgM anti-GRA7 monoclonal antibody TxE2 ( Fischer et al. , 1998 ), rabbit anti-ctag1 antiserum 2600 ( Martens et al. , 2005 ), rabbit anti-calnexin antiserum (StressGene, SPA-865), mouse anti-α-tubulin monoclonal antibody (Sigma-Aldrich, T 6074), mouse anti-Ty-tag monoclonal antibody ( Bastin et al. , 1996 , kindly provided by Keith Gull), rat anti-LAMP1 monoclonal antibody (University of Iowa), anti-Akt (Cell Signaling, 9272) and anti-phosphoAkt (Cell Signaling, 9271) rabbit polyclonal antibodies, rabbit anti-PARP1 polyclonal antibody (Cell Signaling, 9542), Alexa 350/488/546/555/647-labelled donkey anti-mouse, rabbit and goat sera (Molecular Probes), donkey anti-rabbit- (GE Healthcare), donkey anti-goat- (Santa Cruz Biotechnology) and goat anti-mouse-HRP (horseradish peroxidase) (Pierce) antisera.

Techniques: Transfection, Construct, Expressing, Infection, Immunofluorescence, Staining

Journal: Cellular Microbiology

Article Title: Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole

doi: 10.1111/j.1462-5822.2010.01443.x

Figure Lengend Snippet: Accumulation of IRG proteins on the PVM is reduced in virulent T. gondii infection. A. IFNγ-induced MEFs were infected for 2 h with type I virulent (RH and BK), type II (ME49 and NTE) and type III (CTG) avirulent T. gondii strains and assayed microscopically for Irgb6-positive vacuoles (serum A20). Irgb6-positive PVs were counted for each parasite strain from 400–600 intracellular parasites in two independent experiments and pooled. The differences between the avirulent types II and III strains and the virulent type I strains are highly significant by chi-squared test (*** P < 0.001). B. gs3T3 cells were induced with IFNγ and infected with T. gondii ME49 strain (black bars) or RH-YFP strain (white bars). Numbers of Irgb6-, Irgb10-, Irga6- and Irgd-positive PVs were counted in 3–6 experiments for each IRG protein and T. gondii strain and given as a percentage of intracellular parasites. More than 200 intracellular parasites were counted blind per experiment. The mean percentages of positive vacuoles of ME49 or RH-YFP type for each IRG protein are shown. Error bars indicate the standard deviations. The significances of the differences between loading of ME49 and RH-YFP vacuoles are given on the figure (*** P < 0.001, ** P < 0.01, * P < 0.05, by Student's t -test). C. C57BL/6 MEFs were induced with IFNγ and infected with RH-YFP. Irgb6 (blue) and Irga6 (red) were detected in immunofluorescence with serum A20 and mAb 10E7 respectively. Intracellular fluorescent parasites (RH-YFP, green) identified in phase contrast (PhC) are indicated by white arrowheads (strongly IRG-positive) and arrows (weakly IRG-positive). D. gs3T3 fibroblasts were induced with IFNγ and infected with either ME49 or RH-YFP T. gondii strains. Mean fluorescence intensities of Irga6 (serum 165/3) and Irgb6 (serum A20) signals at the PVM were quantified as described in and Experimental procedures . Thirty-five random PVs per data set were quantified blind. For both Irga6 and Irgb6, the different loading intensities on ME49 and RH-YFP vacuoles were highly significant (*** P < 0.001). E. C57BL/6 MEFs were induced with IFNγ and infected with T. gondii RH-YFP strain. Mean fluorescence intensities of Irga6 and Irgb6 were measured for selected PVs expressing no detectable (open circles), weak (grey filled circles) or strong Irga6 staining (black filled circles). The fluorescent intensity profiles of five representative PVs per group are displayed in . F. Photomicrograph of an IFNγ-stimulated MEF shown 2 h after double infection with ME49 strain (indicated by arrowhead) and RH-YFP strain T. gondii (green, indicated by arrow). The ME49 strain parasite shows intense Irgb6 (serum A20, red) accumulation at the PV while the RH-YFP in the same cell has no Irgb6 on the PV. G. IFNγ-stimulated MEFs were infected with T. gondii ME49 strain alone or simultaneously with ME49 and RH-YFP strains. Irgb6 (detected by serum A20) and Irga6 (detected by mAb 10D7) fluorescence intensities were measured on at least 30 ME49 PVs in singly and doubly infected cells. ME49 and RH-YFP were discriminated by the fluorescent signal from RH-YFP. The arithmetic means are given as horizontal lines. The loading of Irga6 and Irgb6 onto PVs of avirulent ME49 strain T. gondii was unaffected by the simultaneous presence of virulent RH-YFP.

Article Snippet: The following immunoreagents were used: rabbit anti-Irga6 antiserum 165/3 ( Martens et al. , 2004 ), mouse anti-Irga6 monoclonal antibodies (mAb) 10E7 and 10D7 ( Papic et al. , 2008 ), mouse anti-Irgm3 monoclonal antibody anti-IGTP (BD Biosciences, 610881), goat anti-Irgb6 antiserum A20 (Santa Cruz Biotechnology, sc-11079), rabbit anti-Irgb6 antiserum 141/1 was raised against recombinant bacterial full-length protein (N. Pawlowski, unpublished data), mouse anti-Irgb6 monoclonal antibody B34 ( Carlow et al. , 1998 ), rabbit anti-Irgm2 antiserum H53/3, raised against the N-terminal peptide of Irgm2 from C57BL/6 and unreactive against Irgm2 from 129 strain mice ( Martens et al. , 2005 and J.C. Howard, unpublished data), rabbit anti-Irgd antiserum 2078/3 ( Martens et al. , 2004 ), rabbit anti-Irgd antiserum 081/1 was raised against bacterially synthesized full-length protein (N. Pawlowski and G. Vopper, unpublished data), rabbit anti-Irgb10 antiserum ( Coers et al. , 2008 ), anti-Irgm1 antiserum L115 B0 ( Martens et al. , 2005 ), anti- T. gondii rabbit (BioGenex, PU125-UPE) and goat antisera (Abcam, ab23507), mouse IgG anti-GRA7 monoclonal antibody ( Bonhomme et al. , 1998 ) and mouse IgM anti-GRA7 monoclonal antibody TxE2 ( Fischer et al. , 1998 ), rabbit anti-ctag1 antiserum 2600 ( Martens et al. , 2005 ), rabbit anti-calnexin antiserum (StressGene, SPA-865), mouse anti-α-tubulin monoclonal antibody (Sigma-Aldrich, T 6074), mouse anti-Ty-tag monoclonal antibody ( Bastin et al. , 1996 , kindly provided by Keith Gull), rat anti-LAMP1 monoclonal antibody (University of Iowa), anti-Akt (Cell Signaling, 9272) and anti-phosphoAkt (Cell Signaling, 9271) rabbit polyclonal antibodies, rabbit anti-PARP1 polyclonal antibody (Cell Signaling, 9542), Alexa 350/488/546/555/647-labelled donkey anti-mouse, rabbit and goat sera (Molecular Probes), donkey anti-rabbit- (GE Healthcare), donkey anti-goat- (Santa Cruz Biotechnology) and goat anti-mouse-HRP (horseradish peroxidase) (Pierce) antisera.

Techniques: Infection, Immunofluorescence, Fluorescence, Expressing, Staining

Journal: Molecules

Article Title: MMI-0100 Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice through Targeting MK2 Pathway

doi: 10.3390/molecules24152832

Figure Lengend Snippet: MMI-0100 prevented DSS-induced colon colitis by activating MK2 signaling pathway. ( A , B ) The production of p-MK2, MK2 was investigated by WB and quantified with statistical significances. Data are presented as mean ± SD. ** p < 0.01 for between DSS group and control group; ### p < 0.001 for between MMI-0100 and DSS group.

Article Snippet: Anti-t-MK2 Rabbit mAb , 1:500 , D16188-0025 , BBI Life Sciences.

Techniques: Control

Journal: Molecules

Article Title: MMI-0100 Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice through Targeting MK2 Pathway

doi: 10.3390/molecules24152832

Figure Lengend Snippet: Antibody information used in this experiment.

Article Snippet: Anti-t-MK2 Rabbit mAb , 1:500 , D16188-0025 , BBI Life Sciences.

Techniques: Concentration Assay

Journal: BMC Research Notes

Article Title: Resveratrol induces H3 and H4K16 deacetylation and H2A.X phosphorylation in Toxoplasma gondii

doi: 10.1186/s13104-020-05416-4

Figure Lengend Snippet: Effect of intracellular exposure to resveratrol (RSV) on T. gondii H2A.X phosphorylation (γH2AX). a Western blot of T. gondii recombinant H2A.X (rH2A.X; 200 ng/lane) expressed in Escherichia coli and purified by nickel resin. Rabbit anti-rTgH2A.X (α-TgH2A.X, 1:5000) and anti- T. gondii phosphorylated peptide (α-TgγH2A.X, 1:100). The phosphorylated peptide sequence was NH - C + GKHGV-S (PO3H2) -QEF -COOH. b Western blot of T. gondii lysates with anti-SAG1 ( T. gondii surface antigen 1, 1:500), anti-TgH2A.X or anti-TgγH2A.X. Lysates were obtained from purified intracellular tachyzoites previously treated with RSV (50 μM) or DMSO 0.5% for 24 h. H2A.X (arrow) and γH2A.X band intensities were quantified and normalized to SAG1 band intensities. The relative intensity of the bands (γH2A.X/H2A.X) was then calculated for each treatment. The signal normalized to SAG1 was calculated from relative values in comparison to the DMSO control. The results are the means of three replicates ± SD. This panel corresponds to Experiment 3 in Additional file . c Quantitation of the fold increase in γH2A.X level after RSV treatment. d Putative model of RSV action that leads to a decrease in T. gondii replication. Dotted lines indicate reduced activities or processes. For acetylations and phosphorylations, color intensity represents the PTM mark level

Article Snippet: A serum sample of rabbit anti- T. gondii γH2A.X was obtained from Eurogentec (Belgium) based on the peptide NH 2 -C + GKHGV-S (PO3H2) -QEF–COOH, designed from the T. gondii H2A.X amino acid sequence.

Techniques: Western Blot, Recombinant, Purification, Sequencing, Quantitation Assay

Journal: Archives of oral biology

Article Title: Treponema denticola upregulates MMP-2 activation in periodontal ligament cells: interplay between epigenetics and periodontal infection

doi: 10.1016/j.archoralbio.2014.06.003

Figure Lengend Snippet: PDL cells were challenged with T. denticola at MOI=100 for 2h, washed twice in PBS and incubated in serum- and antibiotic-free medium with daily changes. Panel A: Gelatinase activity in equal volumes of conditioned medium collected on the indicated days following T. denticola challenge. Panel B: Gelatinase activity in lysates of PDL cells collected on the indicated days following T. denticola challenge and medium replacement as in Panel A. Equal amounts of protein were loaded per lane. The locations of the active dentilisin complex (95-100 kDa), 72-kDa pro-MMP-2 and 64-kDa activated MMP-2 are indicated, as are the positions of relative molecular mass markers in kDa.

Article Snippet: For immunofluorescence microscopy to detect colocalization of T. denticola and LAMP1, PDL cells were treated, challenged and fixed as above, then permeabilized with (0.2% Triton X-100) prior to probing with rabbit anti- T. denticola whole cell IgG and mouse anti-LAMP1 IgG (Enzo Life Sciences, Farmingdale, NY) followed by fluor-conjugated secondary antibodies.

Techniques: Incubation, Activity Assay

Journal: Archives of oral biology

Article Title: Treponema denticola upregulates MMP-2 activation in periodontal ligament cells: interplay between epigenetics and periodontal infection

doi: 10.1016/j.archoralbio.2014.06.003

Figure Lengend Snippet: Panels A and B: T. denticola at MOI=100 was added to PDL cultures for 2h, after which PDL cells were treated (“uptake”) or not treated (“adherence + uptake”) with 200 μg ml-1 gentamicin for 1h to kill extracellular bacteria. After washing and incubation in fresh αMEM for the indicated times, PDL cells were lysed with sterile water, and lysates were mixed with NOS semisolid medium and incubated anaerobically at 37°C. Panel A: T. denticola colony forming units recovered per well of PDL cells (approximately 105 cells) after 0, 7 and 24 h post-challenge incubation. The data represent two independent experiments conducted in triplicate. Panel B: T. denticola colonies recovered from PDL cell lysates in a representative experiment. Panel C: Immunofluorescence microscopy of PDL cells with or without 2h T. denticola challenge (2h, MOI=100) followed by washes, with or without further incubation in culture medium and membrane permeabilization. Slides were probed with rabbit anti-T. denticola Msp IgG followed by Alexa 555-conjugated goat anti-rabbit IgG to detect T. denticola and phalloidin-647 to detect cytoskeletal actin in PDL cells. Panel D: Imunofluorescence microscopy of PDL cells challenged with T. denticola (2h, MOI=100) followed by washes and membrane permeabilization, probed with rabbit anti-T. denticola whole cell IgG and mouse anti-LAMP1 IgG followed by fluor-conjugated secondary antibodies.

Article Snippet: For immunofluorescence microscopy to detect colocalization of T. denticola and LAMP1, PDL cells were treated, challenged and fixed as above, then permeabilized with (0.2% Triton X-100) prior to probing with rabbit anti- T. denticola whole cell IgG and mouse anti-LAMP1 IgG (Enzo Life Sciences, Farmingdale, NY) followed by fluor-conjugated secondary antibodies.

Techniques: Incubation, Immunofluorescence, Microscopy

Journal: Archives of oral biology

Article Title: Treponema denticola upregulates MMP-2 activation in periodontal ligament cells: interplay between epigenetics and periodontal infection

doi: 10.1016/j.archoralbio.2014.06.003

Figure Lengend Snippet: Panels A and B: Transcript levels in PDL cells after T. denticola challenge and incubation in fresh medium for indicated times, assayed by qRT-PCR. The Y-axis in each panel represents fold-expression level of each gene relative to unchallenged control at day 1 shown in Panel A. Panel A: gene expression after 2h T. denticola challenge and incubation in fresh medium for 1, 3 or 5 days. Panel B: gene expression after 4h, 8h or 24h T. denticola challenge and incubation in fresh medium for 24h. Data were analyzed using Student's t-test. Panel C: Western blots showing levels of MT1-MMP expression in PDL cells treated with purified dentilisin (50 ng/ml, 2h) or media control (Ctrl), then washed and maintained in fresh media for 5 days (MT1-MMP1 antibody; Abcam; actin antibody). Panel D: Western blot of TIMP-2 expression in PDL cells (conditioned media) treated for 2h with wildtype T. denticola, T. denticola dentilisin mutant (Td-ΔP) or media control (Ctrl), then washed and maintained in antibiotic supplemented media for 5 days (TIMP-2 antibody, Triple Point Biologics).

Article Snippet: For immunofluorescence microscopy to detect colocalization of T. denticola and LAMP1, PDL cells were treated, challenged and fixed as above, then permeabilized with (0.2% Triton X-100) prior to probing with rabbit anti- T. denticola whole cell IgG and mouse anti-LAMP1 IgG (Enzo Life Sciences, Farmingdale, NY) followed by fluor-conjugated secondary antibodies.

Techniques: Incubation, Quantitative RT-PCR, Expressing, Western Blot, Purification, Mutagenesis

Journal: Archives of oral biology

Article Title: Treponema denticola upregulates MMP-2 activation in periodontal ligament cells: interplay between epigenetics and periodontal infection

doi: 10.1016/j.archoralbio.2014.06.003

Figure Lengend Snippet: PDL cells were challenged with T. denticola for 2h, washed, and incubated in fresh medium for 24 h prior to analysis. The vertical scale indicates percent of total DNA. Panel A: methylation analysis of the MMP-2 promoter region as determined by MethylDNA Restriction Screen (Qiagen). Results are expressed as the percentage of hyper-methylated and unmethylated DNA, which sum to 100%. Data were analyzed using Student's t-test. Panel B: DNA methylation analysis of the MMP-2 promoter region as determined by sequencing of independent PCR clones of bisulfite-converted DNA from T. denticola challenged and unchallenged PDL cell cultures. Each circle represents an individual CpG within residues 236-456 of MMP2-exon 1-transcript variant 1. Open and closed circles indicate unmethylated and methylated CpGs, respectively within residues 236-456 of MMP2-exon 1.

Article Snippet: For immunofluorescence microscopy to detect colocalization of T. denticola and LAMP1, PDL cells were treated, challenged and fixed as above, then permeabilized with (0.2% Triton X-100) prior to probing with rabbit anti- T. denticola whole cell IgG and mouse anti-LAMP1 IgG (Enzo Life Sciences, Farmingdale, NY) followed by fluor-conjugated secondary antibodies.

Techniques: Incubation, Methylation, DNA Methylation Assay, Sequencing, Clone Assay, Variant Assay