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14
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63149183873
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note
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50 > 6.
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-
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15
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63149122988
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note
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2 (5 mM final) in assay buffer (40 mM HEPES pH 7.4, 1 mM DTT) were added to wells containing 1 μl of various concentrations of compound or DMSO vehicle (3% final) in NUNC 384-well black plates. The reaction was initiated by addition of p38α (100 pM final) to give a total volume of 30 μl. After 120 min incubation (rt), 15 μl of 100 mM EDTA pH 7.4 was added followed by detection reagent (15 μl) in buffer (100 mM HEPES pH 7.4, 150 mM NaCl, 0.1% w/v/BSA, 1 mM DTT) containing antiphosphothreonine-ATF2-71 polyclonal antibody (Cell Signalling Technology, Beverly Massachusetts, MA) labelled with W-1024 Eu chelate (Wallac OY, Turku, Finland), and APC-labelled streptavidin (Prozyme, San Leandro, CA). After 60 min further incubation (rt) the ATF-2 phosphorylation was measured using a Packard Discovery plate reader (Perkin-Elmer, Pangbourne, UK) as a ratio of specific 665 nm energy transfer signal to reference Eu 620 nm signal.
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-
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16
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63149113437
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note
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18
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17
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0032528998
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Adams, J.L.1
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20
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63149154322
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note
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TM service, for further details see: www.millipore.com. JNK data were obtained using N-terminal His-tagged full-length human JNK1α1, JNK2α2 or JNK3 with ATF2 as substrate, in the presence of 45, 45, or 10 μM ATP, respectively. Erk-2 data were obtained in a radiometric filter binding assay using N-terminal GST-tagged full-length human Erk-2, activated with MEK1, with myelin basic protein as substrate, in the presence of 155 μM ATP.
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21
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63149165984
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note
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14 was purified following a five stage process after lysis of E. coli cells expressing GST-JNK3t. [Glutathione Sepharose (GSH), Thrombin cleavage, GSH, Source 15-Q anion exchange, SEC]. The protein was supplied in 50 mM Tris/HCl pH 8.0, 150 mM NaCl post final stage Superdex 200 prep grade Size Exclusion column. Co-crystals of Jnk3t with 16 were grown at 20 °C using the hanging drop method combined with micro-seeding. Protein at 13 mg/mL, pre-incubated with 5 mM compound, was mixed with serial dilutions of a Jnk3t seed stock (made in 25% peg 3350, 0.1 M sodium Hepes pH7.5). Drops were then equilibrated over a reservoir containing 18% peg3350, 0.1 M sodium Hepes pH7.5) before freezing in mother liquor plus 15% glycerol. A 2.4 Å dataset was collected from a single frozen crystal of Jnk3t/16 on a Mar345 detector mounted on a micromax 007HF rotating anode generator. The crystal structure was refined starting from the coordinates of another Jnk3t complex crystal stucture (pdb code: 2O0U-ligand removed before start of refinement). The deposition code for the Jnk3t/16 complex is 2waj. Crystal structures of 24 and 26 with a similarly truncated version of Jnk1 are virtually identical (Bax et al., unpublished results).
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24
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33746700144
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Lu Y.-X., Zou J.-W., Wang Y.-H., Zhang H.-X., Yu Q.-S., and Jiang Y.-J. J. Mol. Struct.: THEOCHEM 766 (2006) 119
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26
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-
63149086135
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-
note
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Note that the hydrogen atoms were not resolved in this structure: where necessary for analysis of the H-bond geometries they were added in standard positions using Maestro (Schrödinger Inc.).
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27
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0003914038
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Jeffrey G.A. (Ed), Oxford University Press, New York
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