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21
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44849124311
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50 values were calculated with standard deviation from 2 to 8 separate experiments.
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50 values were calculated with standard deviation from 2 to 8 separate experiments.
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22
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0037676025
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Bhattacharya S.K., Cox E.D., Kath J.C., Mathiowetz A.M., Morris J., Moyer J.D., Pustilnik L.R., Rafidi K., Richter D.T., Su C., and Wessel M.D. Biochem. Biophys. Res. Commun 307 (2003) 267
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Wessel, M.D.11
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23
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0037059757
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Brignola P.S., Lackey K., Kadwell S.H., Hoffman C., Horne E., Carter H.L., Stuart J.D., Blackburn K., Moyer M.B., Alligood K.J., Knight W.B., and Wood E.R. J. Bio. Chem. 277 (2002) 1576
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14C-thymidine incorporation into cellular DNA as described except that total time in which cells were exposed to drug was 96 h
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14C-thymidine incorporation into cellular DNA as described. Emanuel S., et al. Mol. Pharm. 66 (2004) 635 except that total time in which cells were exposed to drug was 96 h
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Emanuel, S.1
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44849109456
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Upstate Cell Signaling Solutions, Charlottesville, VA.
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Upstate Cell Signaling Solutions, Charlottesville, VA.
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26
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44849124962
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PDB code is 3BEL.
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PDB code is 3BEL.
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27
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44849117091
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S671-G998) which was confirmed by DNA sequencing. This expression construct was then integrated into bacmid DNA and a recombinant isolate used to tansfect SF9 insect cells as recommended by the vendor. The resulting baculovirus was then used to infect additional Sf9 cells to increase viral titer and volume.
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S671-G998) which was confirmed by DNA sequencing. This expression construct was then integrated into bacmid DNA and a recombinant isolate used to tansfect SF9 insect cells as recommended by the vendor. The resulting baculovirus was then used to infect additional Sf9 cells to increase viral titer and volume.
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44849104710
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EGFR purification. All purification processes were carried out on an ÄKTA FPLC system (GE Healthcare) at 4 °C. The purification protocol was performed as described previously [J. Biol. Chem. (2002), 277, p. 46265-46272,]. Briefly, frozen cells were thawed and resuspended in 50 mM Tris pH 7.5, 200 mM NaCl, 1% glycerol, 5 mM BME, 1 X complete EDTA-free protease inhibitor cocktail (Roche). Resuspended cells were dounce homogenized and mechanically lysed with an Emulsiflex-C5 (Avestin) at 10,000-15,000 psi. The lysate was clarified by centrifugation at 40,000g for 1 h. The supernatant was filtered through a 0.8 μm vacuum filter and mixed to a TALON metal affinity resin (BD Biosciences) overnight at 4 °C. The lysate-TALON mixture was loaded into a column and washed with 10 column volumes of Buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole.)
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EGFR purification. All purification processes were carried out on an ÄKTA FPLC system (GE Healthcare) at 4 °C. The purification protocol was performed as described previously [J. Biol. Chem. (2002), 277, p. 46265-46272,]. Briefly, frozen cells were thawed and resuspended in 50 mM Tris pH 7.5, 200 mM NaCl, 1% glycerol, 5 mM BME, 1 X complete EDTA-free protease inhibitor cocktail (Roche). Resuspended cells were dounce homogenized and mechanically lysed with an Emulsiflex-C5 (Avestin) at 10,000-15,000 psi. The lysate was clarified by centrifugation at 40,000g for 1 h. The supernatant was filtered through a 0.8 μm vacuum filter and mixed to a TALON metal affinity resin (BD Biosciences) overnight at 4 °C. The lysate-TALON mixture was loaded into a column and washed with 10 column volumes of Buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole.) EGFR was eluted using a 10 column volume linear gradient going from Buffer A to Buffer B (50 mM Tris pH 8.0, 300 mM NaCl, 250 mM Imidazole.) Fractions containing EGFR, as assayed by SDS-PAGE, were pooled. Thrombin was added to the pooled protein to remove the histidine tag (0.005 U of thrombin/μg EGFR). The reaction was dialyzed overnight against 50 mM Tris buffer pH 8.0, 250 mM NaCl, 1 mM DTT. Cleaved EGFR was filtered through a 0.2 μm SFCA cartridge filter, concentrated, and loaded onto a Superdex 200 HR 10/30 column (GE Healthcare,) preequilibrated with 50 mM Tris pH 8.0, 500 mM NaCl, 1 mM DTT. Fractions containing EGFR, as assayed by SDS-PAGE, were pooled and dialyzed against 10 mM Tris pH 8.0, 1 mM DTT, 1 mM sodium azide, 0.1 mM benzamidine.
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44849101004
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16 X-ray diffraction data to a resolution of 2.0 Å were collected at the IMCA-CAT ID-17 beamline at the Argonne National Laboratory. Diffraction data were indexed, integrated, and scaled using the HKL2000 suite. The EGFR crystals belong to the P212121 space group, with unit cell parameters a = 45.50, b = 67.69, c = 103.25 Å. The structure was determined by molecular replacement with CNX [Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., Warren
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16 All model building was done using O [Jones, T. A., Zou, J. Y., Cowan, S. W., Kjeldgaard, M. (1991) Acta Crystallogr A47, 110-119] and refinement and map calculations were carried out using CNX [Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., Warren, G. L. (1998) Acta Crystallogr D 54, 905-921]. The final structure was refined to an Rfactor of 22.7 and Rfree of 26.7.
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0035476866
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Rusnak D.W., Affleck K., Cockerill S.G., Stubberfield C., Harris R., Page M., Smith K.J., Guntrip S.B., Carter M.C., Shaw R.J., Jowett A., Stables J., Topley P., Wood E.R., Brignola P.S., Kadwell S.H., Reep B.R., Mullin R.J., Alligood K.J., Keith B.R., Crosby R.M., Murray D.M., Knight W.B., Gilmer T.M., and Lackey K. Cancer Res. 61 (2001) 7196
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Page, M.6
Smith, K.J.7
Guntrip, S.B.8
Carter, M.C.9
Shaw, R.J.10
Jowett, A.11
Stables, J.12
Topley, P.13
Wood, E.R.14
Brignola, P.S.15
Kadwell, S.H.16
Reep, B.R.17
Mullin, R.J.18
Alligood, K.J.19
Keith, B.R.20
Crosby, R.M.21
Murray, D.M.22
Knight, W.B.23
Gilmer, T.M.24
Lackey, K.25
more..
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Gaul M.D., Guo Y., Affleck K., Cockerill G.S., Gilmer T.M., Griffin R.J., Guntrip S., Keith B.R., Knight W.B., Mullin R.J., Murray D.M., Rusnak D.W., Smith K., Tadepalli S., Wood E.R., and Lackey K. Bioorg. Med. Chem. Lett. 13 (2003) 637
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Griffin, R.J.6
Guntrip, S.7
Keith, B.R.8
Knight, W.B.9
Mullin, R.J.10
Murray, D.M.11
Rusnak, D.W.12
Smith, K.13
Tadepalli, S.14
Wood, E.R.15
Lackey, K.16
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