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Lethal toxin challenge conditions: J774A.1 cells were allowed to grow to confluency in 96-well plates before 3 nM PA, LOPAC 1280 (Sigma) library members or compounds 1-6 (0.6% DMSO), and 0.61 nM LF (List Biological Laboratories) were added to the cells. Cells were allowed to incubate with the PA/LF/compound mixture for 4 h at 37 °C before MTT was added. Cells were allowed to incubate for an additional 1.5 h at 37 °C before cell viability was determined.
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34447324419
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note
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50): compounds 1-6 (0.6% DMSO) were incubated with 50 nM LF (List Biological Laboratories) in assay buffer (65 μL) for 1 h before addition to 4 μM MAPKKide (List Biological Laboratories) in assay buffer (10 μL). Fluorescent measurements were performed over 10 min (excitation and emission of 485 and 590, respectively). Assay buffer consisted of 20 mM Hepes, pH 7.4.
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25
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34447315310
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note
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i): compounds 1, 4 and 6 (1.6% DMSO) were incubated with 12.5 nM LF (List Biological Laboratories) in assay buffer (65 μL) for 1 h before addition to 6-12 μM MAPKKide (List Biological Laboratories) in assay buffer (10 μL). Fluorescent measurements were performed over 10 min (excitation and emission of 485 and 590, respectively). Assay buffer consisted of 20 mM Hepes, pH 7.4.
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0035829518
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Pannifer A.D., Wong T.Y., Schwarzenbacher R., Renatus M., Petosa C., Bienkowska J., Lacy D.B., Collier R.J., Park S., Leppla S.H., Hanna P., and Liddington R.C. Nature 414 (2001) 229
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34447333150
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note
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+).
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30
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34447339418
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note
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+).
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31
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34447333421
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note
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Cytotoxicity study: J774A.1 cells were allowed to grow to confluency in 96-well plates before 0-100 μM compound in media (0.6% DMSO) was added to the cells. Cells were allowed to incubate with the compound mixture for 4 h at 37 °C before MTT was added. Cells were allowed to incubate for an additional 1.5 h at 37 °C before cell viability was determined.
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32
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34447324696
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note
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+).
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