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note
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125I-PrA (Amersham) or by horseradish peroxidase-labeled secondary antibody in conjunction with enhanced chemiluminescence (Amersham) by exposing the filters to Kodak XOMAT film. In some cases, alkaline phosphatase-conjugated protein A and a color reaction mediated by bromochloroindolyl phosphate-nitro blue tetrazcHium (BCIP-NBT) was used.
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19
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1842302603
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note
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Apoptosis was induced by addition of anti-Fas IgM (150 ng/ml; Immunotech) to Jurkat T cells in complete medium for the times indicated. Jurkat T cells were suspended in propidium iodide buffer [0.1 % Na citrate, 0.1% Triton X-100, propidium iodide (20 μg/ml)] and analyzed by fluorescence-activated cell sorting (FACS) as described (29).
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20
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1842280480
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note
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2, 0.05% Triton X-100, and 0.05% Tween 20, the filters were dried and exposed on Hyperfilm (Amersham).
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1842322614
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note
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To obtain active, nonrecombinant caspase fractions, we treated Jurkat T cells with anti-Fas IgM (150 ng/ml; Immunotech) for 3 to 4 hours. At this time point, more than 50% of the cells showed clear signs of apoptosis, such as DNA fragmentation, membrane blebbing, and apoptotic body formation. Cells were washed in ice-cold PBS and lysed in 25 mM Hepes, 0.1% CHAPS, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride, leupeptin (0.5 μg/ml), and aprotinin (10 μg/ml). Cleared lysates were used as active protease fractions in digests of PAK fusion proteins. In general, 300 ng of PAK fusion protein were incubated with 30 μg of either active Jurkat lysate or lysates of E. coli expressing recombinant CPP32 in a total volume of 30 μl of 25 mM Hepes, 1 mM DTT (pH 7.5) at 37°C for 1 hour.
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25
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1842263880
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note
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2+-nitrilotriacetic acid resin (Qiagen).
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T. Rudel and G. M. Bokoch, data not shown
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T. Rudel and G. M. Bokoch, data not shown.
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27
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1842272641
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note
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Complementary DNA encoding CPP32 was cloned in-frame into the Bam Hl-Eco Rl site of pGEX-4T-3. Expression of CPP32 and preparation of active bacterial lysates has been described (25); expression was induced in exponentially growing bacteria by addition of 1 mM IPTG for 3 hours at 37°C.
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212 of PAK2 (Glu-Val-Asp-Gly-Ala) compares favorably with that of the known CPP32 cleavage site in poly-(ADP ribose) polymerase (His-Val-Asp-Gly-Ile).
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31
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1842278496
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note
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32P]ATP (adenosine 5′-triphosphate) per milliliter (5000 Ci/mmol) and 20 μM ATP for 30 min at 30°C. The gels were washed several times in 5% (w/v) trichloroacetic acid and 1% sodium pyrophosphate, stained with Coomassie blue, and dried for autoradiography.
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1842311097
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note
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86 mutations in the GTPase binding site prevents Rac or Cdc42 binding (7), which allowed us to avoid potentially complicating effects resulting from titration of Rac or Cdc42.
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34
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1842276583
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note
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7 Jurkat T cells, which were already stably transfected with the lacl-encoding p3′SS vector (Stratagene), and electroporated. Transfected cells were cultivated for 24 hours before addition of G418 (1 mg/ml; Gibco-BRL) to select for transfectant clones. To ensure the clonal origin, transfectants were aliquoted in serial dilutions into 96-well culture plates and expanded only from plates with less than 10 G418-resistant clones per 96-well plate.
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1842315816
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2. The cells were washed in PBS, fixed in 3.8% formaldehyde, and air-dried on microscope slides. Nicked and degraded DNA was labeled by use of the terminal deoxynucleotidyl transferase (TdT) reaction with digoxygenin-labeled nucleotides and fluorescein-conjugated anti-digoxygenin. The samples were mounted with Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL) and viewed under a Nikon Labophot-2 microscope at 100× magnification.
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1842353513
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note
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Exposure of PS was quantified by annexin-V-FITC binding and flow cytometry with the Apoptosis Detection Kit (R&D Systems). Jurkat T cells and mutant cell line TA12 were incubated with anti-Fas IgM for the indicated time, washed with PBS, and resuspended in binding buffer. Annexin-V-FITC and Pl were added, and the samples were analyzed by flow cytometry in a FACScan (Becton Dickinson). Annexin-V-FITC was measured in the FL1 channel, and Pl was measured in the FL3 channel. Annexin-V-FITC binding positive-staining cells and Pl negative-staining cells were scored as apoptotic.
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45
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1842305394
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note
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We thank T.-H. Chuang for assistance with Jurkat transfection procedures and for scientific advice. Supported by the Deutsche Forschungsgemeinschaft (T.R.) and NIH grants HL48008 and GM39434 (G.M.B.). This is publication number 10589-IMM from The Scripps Research Institute.
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