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. 2007 Dec 5;82(4):2004–2012. doi: 10.1128/JVI.02017-07

FIG. 2.

FIG. 2.

FHV RNA replication in S. cerevisiae is independent of Hsp90 chaperone complex activity. (A) Inducible Hsp90-dependent GR activity. Wild-type (black bars), Δsti1 (white bars), and Δsba1 (gray bars) yeast cells transformed with pSC-GRE-LacZ and pSC-FLGR were induced with 10 μM deoxycorticosterone (DOC) in the presence or absence of 10 μM geldanamycin (GA), and β-galactosidase activity was measured in a colorimetric assay. Results are expressed as the percent activity relative to DOC-induced β-galactosidase in wt yeast. (B) Constitutive Hsp90-independent GR activity. Wild-type (black bars), Δsti1 (white bars), and Δsba1 (gray bars) yeast cells transformed with pSC-GRE-LacZ and pSC-cGR were assayed for β-galactosidase activity, and results are expressed as the percent activity relative to wt yeast. (C) FHV RNA replication in Δsti1 yeast. Yeast transformed with the vector control (lanes 1 and 4) or pF1 (lanes 2, 3, 5, and 6) were induced with galactose for 24 h, and total protein and RNA were isolated and analyzed by immunoblotting and Northern blotting, respectively. The positions of FHV genomic and subgenomic positive-sense RNA [(+)RNA1 and (+)RNA3, respectively] are shown on the left. rRNA and PGK are shown as loading controls. (D) FHV RNA replication in Δsba1 yeast. Yeast cells transformed with vector control (lanes 1 and 6) or pF1 (lanes 2 to 5 and 7 to 10) were induced with galactose for 24 h in the presence of vehicle (lanes 1 to 3 and 6 to 8) or 10 μM GA (lanes 4, 5, 9, and 10), and total protein and RNA were isolated and analyzed by immunoblotting and Northern blotting, respectively. Labels and loading controls are as described for panel C. Numbers represent the percent accumulation of (+)RNA3 and protein A (PtnA) in Δsti1 or Δsba1 samples compared to the wt control (lanes 2 and 3). There was no difference in growth between wt, Δsti1, and Δsba1 yeast cells after induction with galactose (data not shown). When cropping was necessary, all panels were from the same exposure of the blot, and all contrast adjustments to the initial image were done prior to cropping.