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| | Kinase Assay |
| Description: | Pharmacol 2002, 62 58-62) The compounds named in the specified Examples were tested as follows for inhibition of ALK-5 autophosphorylation activity and of the ALK-5 phosphorylation of α-Casem Materials. Buffer 50 mM HEPES, pH 76, with 10 mM NaCI, 10 mM MgCI2, and 1 mM DTT GST-ALK-5 protein - 0 44 mg/ml (roughly 7 μM stock) A 1 350 dilution gives a 20 nM stock, which translates to 2 nM final in assay Human ALK-5 was expressed in Sf9 insect cells infected with Bacul ovirus expressing a ALK-5 truncation sequence (amino acids H149 -M503), fused at the N-terminus to Glutathione S-transferase GST, in a pFastBac vector (Invitrogen) The cells were disrupted by sonication at 4 0C The lysate was centrifuged at 40,000 x g for 45 minutes, and the supernantant applied to a 10 ml column of Glutathione Sepharose 4 Fast Flow (AmershamBioscienses) equilibrated with 100 mM Tπs-HCI pH 76 buffer containing 300 mM NaCI, 10% glycerol, 1 % NP40, 2 mM dithiothreitol (DTT) and one Protease... |
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