Journal: Journal of Biological Chemistry

Article Title: Loading of the Nonhomologous End Joining Factor, Ku, on Protein-occluded DNA Ends

doi: 10.1074/jbc.m611125200

Figure Lengend Snippet: FIGURE 7. The DNA binding activity of Ku at RAG bound signal ends. A, a signal end complex is defined here as RAG1, RAG2, and HMG1 bound to two- oligonucleotide DNA duplexes, one containing the 12-RS and the other con- taining the 23-RS. A streptavidin-biotin complex was used to block the ends of the DNA duplexes distal to the site of cleavage. B, approximately 10 ng/l of purified RAG1 and RAG2 and 425 nM HMG1 were incubated with 0.4 nM radiolabeled23-RS-containingand10nM12-RS-containingDNAfragmentsat 37 °C for 10 min. 5 M streptavidin was added at 25 °C for 5 min prior to addition of 25 nM Ku. Antibody supershifts required an additional 10-min room temperature incubation step with either 0.2 g of the monoclonal anti- body to Ku or 1 l of a polyclonal antisera recognizing the maltose binding domain fused to recombinant RAG proteins. The inferred composition of each of the generated species is indicated to the side of the panel: species I, 23-RS; species II, HMG1-bound 23-RS; species III, RAGs and HMG1 bound to the 23-RS; species IV, SEC; species V, streptavidin-blocked SEC (upper arrow indi- cates -MBP supershift); species VI, Ku bound to incompletely formed SEC (upper arrow indicates -Ku supershift).

Article Snippet: The relative amounts of Ku and histoneH3 in each of the excised complexes were determined by semi- quantitative Western analysis probing with a polyclonal rabbit antibody raised against native, recombinant human Ku and a polyclonal antibody against histone H3 (Ab1791; Abcam) using fluorescent detection and a Typhoon imager (GE Healthcare).

Techniques: Binding Assay, Activity Assay, Blocking Assay, Purification, Incubation, Recombinant, Generated

Journal: PLoS ONE

Article Title: Networked T Cell Death following Macrophage Infection by Mycobacterium tuberculosis

doi: 10.1371/journal.pone.0038488

Figure Lengend Snippet: (A) Addition of the pan-caspase inhibitor Z-VAD-FMK mitigated cell death induced by low MOI and high MOI-infected macrophage supernatants (shaded bars, infection + and ++ respectively), as well as background cell death observed in the uninfected control supernatants (closed bars, infection -). Cell death in infected supernatants was not mitigated by coincubation with blocking antibodies to TNF-α (B) or Fas (C), and in each case was significantly higher than in uninfected control supernatants (closed bars). Cell death induced by positive controls (D) (1 µM Staurosporine, 5 ng/ml recombinant human TNF-α +0.2 µg/ml cycloheximide and 10 ng/ml recombinant human Fas ligand) was in each case significantly reduced by coincubation with the corresponding inhibitor (open bars). Data shown in each panel are from one representative experiment, showing mean percentage cell death ± SEM from triplicate incubations. ** = p<0.01, *** = p<0.001, Student’s t test relative to uninfected control (A–C), or positive control relative to inhibitor (D).

Article Snippet: In positive control experiments for Fas ligand-mediated cell killing, cells were incubated in the presence of 10 ng/ml recombinant human Fas ligand (R&D systems), crosslinked with 10 μg/ml anti-6× Histidine (R&D systems).

Techniques: Infection, Control, Blocking Assay, Recombinant, Positive Control