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i = dissociation constant for inhibitor binding) of a potent inhibitor the anisotropy of the unbound fluorescent ligand is measurably different from the bound value. Truncated human JNK3 was expressed in baculovirus as a N-terminal His (6)-tagged fusion protein.[...].
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i = dissociation constant for inhibitor binding) of a potent inhibitor the anisotropy of the unbound fluorescent ligand is measurably different from the bound value. Truncated human JNK3 was expressed in baculovirus as a N-terminal His (6)-tagged fusion protein.[...].
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11 p38α inhibitory activity data were generated using conditions previously described, see: Barker, M. D.; Hamblin, J. N.; Jones, K. L.; Patel, V. K.; Swanson, S.; Walker, A. L. Int. Patent Appl. WO 05/073232.
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14
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ERK2 kinase inhibitory activity was determined in a similar fashion to that described above for JNK3, using recombinant full length ERK2 expressed and purified as previously described, see: The ERK2 used for the work described in this paper was not activated before or during the assay
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ERK2 kinase inhibitory activity was determined in a similar fashion to that described above for JNK3, using recombinant full length ERK2 expressed and purified as previously described, see:. McDonald O.B., Chen W.J., Ellis B., Hoffman C., Overton L., Rink M., Smith A., Marshall C.J., and Wood E.R. Anal. Biochem. 268 (1999) 318 The ERK2 used for the work described in this paper was not activated before or during the assay
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33846949470
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2 and 1 mM AMP-PNP. The crystals were grown at 10 °C, using the hanging drop vapour diffusion method, with a reservoir solution of 50 mM Tris/HCl, pH 7.2-7.6, 18-22% PEG3350 and 0.2 M KF and 1 μl + 1 μl hanging drops. JNK3t+MgAMP-PNP cocrystals were soaked at 20 °C in 50 mM Tris/HCl, pH 7.5, 20% PEG3350, and 0.2 M KF containing 0.8 mM inhibitor 5e for 4 days. The 8a complex was obtained in a similar way though crystals were soaked for 1 day in buffer (10 μL) containing 0.1 mg of solid 8a with 1 mM EDTA. Each crystal was cryoprotected by brief immersion into paratone-N (Hampton Research) prior to data collection. The PDB deposition codes for the 5e and 8a JNK3 complex crystal structures are 2O2U and 2O0U, respectively.
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The ability of 5c at 10 μM concentration to displace the fusion protein from immobilised ATP-site probe ligands was determined as previously described, see:
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The ability of 5c at 10 μM concentration to displace the fusion protein from immobilised ATP-site probe ligands was determined as previously described, see:. Fabian M.A., Biggs III W.H., Treiber D.K., Atteridge C.E., Azimioara M.D., Benedetti M.G., Carter T.A., Ciceri P., Edeen P.T., Floyd M., Ford J.M., Galvin M., Gerlach J.L., Grotzfeld R.M., Herrgard S., Insko D.E., Insko M.A., Lai A.G., Lélias J.-M., Mehta S.A., Milanov Z.V., Velasco A.M., Wodicka L.M., Patel H.K., Zarrinkar P.P., and Lockhart D.J. Nat. Biotechnol. 23 (2005) 329
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more..
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33846963281
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note
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33P]ATP (45 μM). For further details, see: www.upstate.com.
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