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ATP, and inhibitors with a 6 min. incubation time. The
degree of phosphorylation was then measured with HRP
conjugated-PY-54 antiphosphotyrosine antibody. The
colorimetric signal was then developed with TMB micro-
well peroxidase substrate and the absorbance was mea-
sured at 450 nm.
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16. erbB2 and EGFR cell assays were conducted as described
in Refs. 9,10. Briefly, NIH3T3 cells transfected with either
human EGFR or a chimeric receptor with EGFR extra-
cellular domain and erbB2 intracellular domain were
seeded in a 96-well tissue culture plate. Inhibitors were
added and incubated for 2 h. Cells were stimulated with
human recombinant EGF for 15 min. The degree of
phosphorylation was then measured with HRP conju-
gated-PY-54 antiphosphotyrosine antibody. The colori-
metric signal was then developed with TMB microwell
peroxidase substrate and the absorbance was measured at
450 nm.
17. EGFR kinase selectivity was not measured routinely
because it is thought that the cellular system is more
biologically relevant with both cell assays performed
under identical conditions, and because EGFR kinase
and cell selectivity have correlated well in previous
studies.9,10 Examples in this publication include: 15f:
kinase sel. = 6·, cell sel. = 23·; 15i: kinase sel. = 178·,
cell sel. = >300·; 21e: kinase sel. = >480·, cell
sel. = >410·.
18. Moyer, J. D.; Barbacci, E. G.; Iwata, K. K.; Arnold, L.;
Boman, B.; Cunningham, A.; Diorio, C.; Doty, J.; Morin,
M. J.; Moyer, M. P.; Neveu, M.; Pollack, V. A.; Pustilnik,
L. R.; Reynolds, M. M.; Sloan, D.; Theleman, A.; Miller,
P. Cancer Res. 1997, 57, 4838.
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J.; Frost, H. N.; Hawkins, J.; Johnson, P. J.; Massett, S.
S.; Neumann, K.; Phillips, J.; Raggon, J. W.; Rose, P. R.;
Rutherford, J. L.; Sitter, B.; Stewart, A. M., III; Vetelino,
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15. erbB2 kinase assays were conducted as described in Refs.
9,10. Briefly, recombinant erbB intracellular domain
(residues 675–1255) was expressed in Baculovirus-infected
Sf9 cells as GST fusion proteins and purified. The
phosphorylation of poly(Glu, Tyr) was then measured in
the presence of ꢀ15 ng of this recombinant protein,1 mM
20. Pouton, C. W. Adv. Drug Deliv. Rev. 1997, 25, 47.