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later. After addition of compound, cells were allowed to grow for either an
additional 72 or 144 h, depending on rate of growth. At harvest, media was
removed and DNA content for individual wells was determined using
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antibody on cells treated for 24 h with compound followed by methanol
fixation. High Content Analysis (HCA) was done using an ArrayScan 4.5
(Pittsburg, PA) or Becton Dickenson 435 (Rockville, MD) imager on cells
treated 24 h with compound. After fixation in 4% PBS-buffered formalin,
cells were probed with anti-Her2 (Millipore), anti-phospho-S6 (pS6) (Cell
Signaling) and anti-Hsp70 (Assay Design) primary antibodies, followed by
TRITC or FITC conjugated secondary antibodies.
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12. An untagged N-terminal domain construct comprising residues 1–232 of
Hsp90 was co-crystallized with compound 16. Amino acids 16–224 and
281 water molecules were located. Number reflections = 27945;
% completeness = 95; RCryst = 0.200; Rfree (5%) = 0.228; resolution (Å) = 1.74–
66.52; space group = I222; unit cell (Å): a = 66.85, b = 90.60, c = 98.22, a = 90°,
b = 90°, g = 90°. The PDB code is 3D0B.
13. Test compound affinity for Hsp90 was determined as follows: Hsp90 from porcine
spleen extract was isolated by affinity capture on a purine-affinity media. The
Hsp90 loaded media was then challenged with test compound at a given
concentration, ranging from 0.8 to 500 lM, and the amount of Hsp90 liberated
at each concentration was determined by Bradford protein assay. The resulting
IC50s were corrected for the ATP ligand concentration and presented as Kd values.
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