Journal: The Journal of Experimental Medicine

Article Title: CD28-independent, TRAF2-dependent Costimulation of Resting T Cells by 4-1BB Ligand

doi:

Figure Lengend Snippet: Association between the 4-1BB cytoplasmic tail-GST fusion protein and TRAF1 and TRAF2 proteins. As described in Materials and Methods, purified GST, GST-CD30CT, and GST–4-1BBCT proteins bound to glutathione beads were incubated with 35 S-labeled in vitro– translated TRAF1 or TRAF2. After washing, samples were eluted and analyzed by SDS-PAGE followed by autoradiography. In vitro–translated TRAF1 and TRAF2 were also run directly on the gel (lanes marked TRAF1 and TRAF2) for comparison.

Article Snippet: For in vitro association experiments, the full-length murine TRAF1 and TRAF2 cDNA in pBluescript (Stratagene Inc., La Jolla, CA) were transcribed and translated in vitro using the TNT coupled reticulocytes system ( Promega ) with 35 S-labeled methionine.

Techniques: Purification, Incubation, Labeling, In Vitro, SDS Page, Autoradiography

Journal: The Journal of Experimental Medicine

Article Title: CD28-independent, TRAF2-dependent Costimulation of Resting T Cells by 4-1BB Ligand

doi:

Figure Lengend Snippet: Association of TRAF1 and TRAF2 with 4-1BB in a T cell hybrid is induced by receptor aggregation at 37°C. C8.A3 T cells (2 × 10 7 cells/ lane) were stimulated with anti– 4-1BB or with anti–ICAM-1 on ice for 5 min and then cross-linked with anti–rat Ig for 15 min on ice or at 37°C. Lysates were immunoprecipitated with protein G–Sepharose. Immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting with either anti-TRAF1 or anti-TRAF2. This experiment is representative of three separate experiments.

Article Snippet: For in vitro association experiments, the full-length murine TRAF1 and TRAF2 cDNA in pBluescript (Stratagene Inc., La Jolla, CA) were transcribed and translated in vitro using the TNT coupled reticulocytes system ( Promega ) with 35 S-labeled methionine.

Techniques: Immunoprecipitation, SDS Page, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: CD28-independent, TRAF2-dependent Costimulation of Resting T Cells by 4-1BB Ligand

doi:

Figure Lengend Snippet: Decreased response to 4-1BBL by T cells isolated from mice expressing a dominant negative form of TRAF2. Resting T cells were isolated from spleen and lymph nodes TRAF2 DN mice or their transgene-negative littermates, as described in Materials and Methods. T cells were incubated with anti-CD3 immobilized on plastic at the concentrations indicated in the figures in the presence or absence of immobilized s4-1BBL (5 μg/ml) or immobilized anti-CD28 at 2 or 10 μg/ml as indicated in the figure. At 48 h of culture, supernatants were analyzed for IL-2 production using CTLL cells. Results shown are for a single representative mouse. Similar results were obtained in three independent experiments with two control and two mutant mice analyzed separately in each experiment.

Article Snippet: For in vitro association experiments, the full-length murine TRAF1 and TRAF2 cDNA in pBluescript (Stratagene Inc., La Jolla, CA) were transcribed and translated in vitro using the TNT coupled reticulocytes system ( Promega ) with 35 S-labeled methionine.

Techniques: Isolation, Expressing, Dominant Negative Mutation, Incubation, Mutagenesis

Journal: The Journal of Experimental Medicine

Article Title: CD28-independent, TRAF2-dependent Costimulation of Resting T Cells by 4-1BB Ligand

doi:

Figure Lengend Snippet: Decreased response to 4-1BBL by T cells isolated from TRAF2 − mice. Resting T cells were isolated from the lymph nodes of TRAF2 − mice or their wild-type TRAF2 + littermates. T cells were incubated with anti-CD3 alone (immobilized at 1 μg/ml) or with anti-CD3 plus immobilized s4-1BBL (5 μg/ml) or immobilized anti-CD28 (2 μg/ml). Results are shown from one mouse and are representative of two independent experiments with two mice per experiment.

Article Snippet: For in vitro association experiments, the full-length murine TRAF1 and TRAF2 cDNA in pBluescript (Stratagene Inc., La Jolla, CA) were transcribed and translated in vitro using the TNT coupled reticulocytes system ( Promega ) with 35 S-labeled methionine.

Techniques: Isolation, Incubation