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. 2009 Sep 8;7(9):e1000188. doi: 10.1371/journal.pbio.1000188

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Huang et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Pho85 regulates Pcl9 protein stability. Wt (BY263) and pho85Δ strains (BY391) harboring a GAL1-PCL9^HA plasmid (pBA2112) were grown to exponential phase in galactose media (lane 1). PCL9 expression was repressed by addition of glucose to final concentration of 2% and cells were harvested 10 (lane 2), 30 (lane 3), and 90 (lane 4) min after addition of glucose. Pcl9 abundance was assessed by immunoblotting using 12CA5 anti-HA antibodies. (B) Pcl9 localizes to SBF-dependent promoters. An exponentially growing GAL1-CDC20 pho85ΔPCL9^MYCstrain (BY4148, lane 1) was arrested at M/G1 phase in glucose-containing medium (lane 2). Cultures were harvested 15 (lane 3), 30 (lane 4), 45 (lane 5), and 60 (lane 6) min after release from CDC20-induced arrest in galactose medium. Cell cycle progression was monitored by FACS analysis. Anti-MYC and anti-Swi6 ChIPs from the indicated strains were analyzed for CLN2 promoter sequences by quantitative RT-PCR. (C) In a strain lacking Whi5, GAL1-CDC20 pho85Δ whi5ΔPCL9^MYC, Pcl9 no longer localizes to the CLN2 promoter. Anti-Swi4 ChIPs are shown as a positive control.