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Volumn 276, Issue 5318, 1997, Pages 1571-1574

Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2

Author keywords

[No Author keywords available]

Indexed keywords

GUANOSINE TRIPHOSPHATASE;

EID: 0030918572     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.276.5318.1571     Document Type: Article
Times cited : (609)

References (45)
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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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    • 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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    • 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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    • 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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