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. 2022 Apr 12;13:1968. doi: 10.1038/s41467-022-29591-z

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Promoter activity of PDR5 in WT and rtt106Δ assessed by luciferase assay. Data are presented as mean values ± SD (n = 3 biological replicates). Statistical analysis performed by an unpaired t-test (two-tailed). b Western blot analysis of Pdr5 in WT and rtt106Δ. HA-tagged Pdr5 expressed from its endogenous locus was detected by anti-HA antibody. For loading control, whole proteins were detected by the Stain-free technology. c Sensitivity of WT, rtt106Δ and pdr5Δ to azole antifungal drugs. Five-fold dilutions of WT, rtt106Δ and pdr5Δ cells were spotted on YPD and YPD containing ketoconazole or fluconazole and incubated at 30 °C for 3 days. d ChIP-qPCR analysis testing if Rtt106 binding at the PDR5 promoter is dependent on Pdr1 and/or Pdr3. ChIP efficiency, the recovery of ChIPed DNA relative to the amount of input. Data are presented as mean values ± SD (n = 3 biological replicates). Statistical significance determined by one-way ANOVA with post-hoc Tukey HSD test. ns, P > 0.05. e GST pull-down analysis shows GST-Pdr3 interacts with Rtt106-6HA. Yeast cell lysate containing Rtt106-6HA was incubated with GST-Pdr3 bound to glutathione-Sepharose beads. WB western blot. f ChIP-qPCR analyses assessing dependence of Rtt106 binding to PDR5 (grey) and HTA1-HTB1 (white) promoters on Pdr1, Pdr3, Hir1, Asf1, Rtt109 and Yta7. ChIP efficiencies relative to WT are shown. Data are presented as mean values ± SD (n = 3 biological replicates). ns, P > 0.05, one-way ANOVA with post-hoc Tukey HSD tests. g Northern blot analysis of PDR5 mRNA in indicated strain backgrounds in YPD. Intensities relative to PDR5 mRNA in WT are shown below the PDR5 blot. rRNAs, loading control, detected by SYBR Gold. The positions of 25 S and 18 S rRNAs are shown. h Sensitivity to ketoconazole of indicated mutant combinations. Five-fold dilutions of indicated strains were spotted on YPD and YPD containing ketoconazole and incubated at 30 °C for 3 days. Two independent isolates of pdr1-3 rtt106Δ and pdr3-2 rtt106Δ strains shown.