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PP1c was purchased from New England Biolabs, and PP2Ac was purchased from Sigma. Protein phosphatase activity was assayed at 37°C using 0.025 units/μL of protein and 100 μM of fluorescein diphosphate as substrate. The reaction was followed by fluorescence spectroscopy at 485-535 nM. Data were fitted to a one-site binding model. Assays with 19-epi-okadaic acid were performed in quintuplicate (n = 5), while for okadaic acid only duplicates (n = 2) were used.
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PP1c was purchased from New England Biolabs, and PP2Ac was purchased from Sigma. Protein phosphatase activity was assayed at 37°C using 0.025 units/μL of protein and 100 μM of fluorescein diphosphate as substrate. The reaction was followed by fluorescence spectroscopy at 485-535 nM. Data were fitted to a one-site binding model. Assays with 19-epi-okadaic acid were performed in quintuplicate (n = 5), while for okadaic acid only duplicates (n = 2) were used.
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0037061628
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Assays adding BSA (0.125 mg/mL) to the media were also undertaken in order to explore the possibility that 19-epi-OA could be a phony hit as described in McGovern, S. L.; Caselli, E.; Grigorieff, N.; Shoichet, B. K. J. Med. Chem. 2002, 45, 1712-1722, but no significant differences were seen, leaving out such possibility.
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Assays adding BSA (0.125 mg/mL) to the media were also undertaken in order to explore the possibility that 19-epi-OA could be a "phony" hit as described in McGovern, S. L.; Caselli, E.; Grigorieff, N.; Shoichet, B. K. J. Med. Chem. 2002, 45, 1712-1722, but no significant differences were seen, leaving out such possibility.
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