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note
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6 irradiated (2000 rad) and T cell-depleted syngeneic splenocytes plus ovalbumin in 96-well culture plates (Costar) at 37°C At 20 hours, supernatant was removed and IL-2 and IL-4 were measured by enzyme-linked immunosorbent assay (ELISA) (Endogen) Anergic cells were rested in culture medium for 2 to 6 days before restimulation Control cells were stimulated with antigen-APC for 7 days, after which they were treated identically to anergized cells.
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12
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13344285514
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note
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4, leupeptin (10 μg/ml), aprotinin (10 μg/ml). 25 mM p-nitrophenyl p-guanidinebenzoate, 1.0 mM phenylmethylsulfonyl fluoride (PMSF), and 10 mM NaF (pH 7.4) (buffer 1). Proteins were immunoprecipitated from post-nuclear lysates with antibody-coated agarose beads and resolved by electrophoresis and immunoblotting. For She and Grb-2-mSOS determinations, cells were lysed in buffer 1 and immunoprecipitation and resolution of proteins were carned out as described above
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13
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13344278856
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note
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Cells were stimulated by soluble cross-linking of CD3 and CD4 with the use of mAbs GK1 5 and 145-2C11 (1:1 mixture) (23) After precoating of cells with this mAb cocktail, 0.5 ml of cross-linking antisera goat antiserum to hamster [(20 μg/ml) (Cappel)] was added and cells were stimulated while being mixed on a rotator at 37°C. This antibody bound to both GK1.5 and 145-2C11 efficiently (7) At various times, cells were pelleted by centrifugation (10,000g for 5 s) for lysis.
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13344283567
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note
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2, bovine serum albumin (1 mg/ml), 0.1 mM GTP, 0.1 mM GDP, 1 mM adenosine triphosphate, 1 mM sodium phosphate (pH 7.4), 0.4 mM PMSF, aprotinin (10 μg/ml), soybean trypsin inhibitor (10 μg/ml), and 10 mM benzamidine. Ras was immunoprecipitated with the mAb Y13-259 (American Type Tissue Collection). Nucleotides were eluted from Ras, separated by thin-layer chromatography, and quantified by direct scanning for beta emissions (AMBIS Systems, San Diego, CA;. Ras immunoblot analysis showed that similar amounts of protein were immunoprecipitated from both control and anergic cells (7). Data are expressed as the ratio of GTP bound to Ras to total guanine nucleotide bound to Ras.
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0025193117
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13344268419
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note
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IL-2 production by antigen-stimulated control and anergic cells was 3969 pg/ml and 33 pg/ml, respectively; whereas control and anergic cells stimulated with PMA (100 ng/ml) + 1 0 μM ionomycin produced IL-2 amounts of 1561 pg/ml and 791 pg/ml, respectively. Similar results were obtained in three separate experiments. The data must be interpreted cautiously, because stimulation of T cells with these agents may not precisely mimic TCR stimulation. Although PMA + ionomycin equivalently activated Ras in control and anergic cells, there was an incomplete recovery of functional responses in the anergic clones, which suggests that other Ras-independent pathways may be affected in anergy.
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23
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13344262163
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note
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Immunoblot analysis revealed no differences in expression of She, Grb-2, or mSOS in control and anergic cells (7).
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γ ELISA reagents.
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