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All strains used in this study were derived by transformation and crosses between the isogenic strains W303a and W303α. The sti1Δ strain was made congenic with W303a through repeated backcrosses.
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10544246348
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note
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Because the growth rate of cells originating from a different strain background (S288C) was less affected by deletion of CPR7, the magnitude of the effect of a cpr7Δ mutation on Hsp90-dependent events may vary from strain to strain.
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40
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10544229628
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note
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2O to a final volume of 200 μl. After incubation at 4°C for 1 hour, the beads were washed four times with 1× binding buffer, and bound proteins were eluted with 0.5× final concentration of glutathione elution buffer (4). Material from 10 μl of each eluate (from a total of 50 μl) was resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and analyzed as described in the figure legend.
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41
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10544256024
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note
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4-terminus of Cpr1, a hybrid between GST and the TPR-containing COOH-terminal region of Cpr6 (amino acids 171 to 371, Cpr6 tail), and a fusion between GST and the analogous region of Cpr7 (amino acids 193 to 393, Cpr7 tail). The binding reactions were treated as described (28), and eluates were analyzed by SDS-PAGE. Immunoblot experiments verified the identity of Hsp90 found in association with the fusion proteins.
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10544238742
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note
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Cells were transformed with a GR expression plasmid, p2A/GRGZ, that also contains the lacZ gene under the control of GR elements (8). In the absence of the receptor, the lacZ reporter gene is expressed at very low levels (14). Mid-logarithmic phase cells grown in galactose-containing medium were treated with saturating amounts (20 μM) of deoxycorticosterone (DOC) for 1 hour at 25°C, and β-galactosidase activity was assessed as described (8). Cellular proteins from mid-logarithmic phase cells were separated on an SDS-polyacrylamide gel and transferred to an Immobilion-P membrane. Equal sample loading was confirmed by Coomassie blue staining before incubation with antibody specific for GR (8) or L3 proteins. Constitutive GR activity was measured without DOC (8).
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43
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10544236407
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note
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We thank I. Yahara for plasmid pETy90, J. Warner for antibody to L3 protein, and K. Yamamoto for reporter and constitutive GR-expressing plasmids (pSX26.1 and pGN556). We also thank J. Heitman for communicating unpublished results regarding wild-type amounts of Hsp90-dependent GR activity in cpr6Δ cells, and M. H. Vos for technical assistance. Supported by NIH grant GM45739 (R.F.G.) and GM25874 (S.L.).
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