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. 2019 Nov 20;10:5237. doi: 10.1038/s41467-019-13243-w

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© The Author(s) 2019

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. 3 — TTT and Hsp90 promote de novo assembly of Tra1 into SAGA. a Silver staining analysis of SAGA complexes purified upon Tra1 synthesis (neo-Tra1). spt7-TAP RI-tra1 cells were grown to exponential phase and harvested at different time points after β-estradiol addition, as indicated (hours). SAGA was purified from a WT strain as a positive control, in which the asterisk labels steady-state Tra1 levels. Below is an anti-HA western blot of Spt7-TAP from a fraction of the input used for TAP. Ponceau red staining is used as loading control. Data are representative of four independent experiments. b Silver staining of SAGA complexes purified upon Tra1 synthesis (neo-Tra1), either in presence or absence of the TTT subunit Tel2. spt7-TAP RI-tra1 tel2-AID cells were grown to exponential phase in rich medium, supplemented with either ethanol (−IAA) or auxin (+IAA) for 16 h, and harvested 6 h after addition of either DMSO (−) or β-estradiol (+). SAGA was purified from a WT strain as a positive control, in which the asterisk labels steady-state Tra1 levels. Below are anti-HA western blotting of Spt7-TAP and Tel2-AID from a fraction of the input used for TAP. Both the TAP and AID sequences are in frame with HA epitopes. Ponceau red staining is used as loading control. Data are representative of two independent experiments. c Silver staining of SAGA complexes purified upon Tra1 synthesis (neo-Tra1), either in WT or hsp90–201 mutant strains. spt7-TAP RI-tra1 hsp90 and spt7-TAP RI-tra1 hsp90–201 cells were grown to exponential phase in rich medium and harvested 6 h after addition of either DMSO (−) or β-estradiol (+). Below is an anti-HA Western blot of Spt7-TAP from a fraction of the input used for TAP. Ponceau red staining is used as loading control. Data are representative of two independent experiments. d LC-MS/MS analyses of SAGA purifications from control spt7-TAP RI-tra1 strains, spt7-TAP RI-tra1 tel2-AID strains used in (d), and spt7-TAP RI-tra1 hsp90–201 strains used in (e). LFQ intensity ratios of newly synthesised Tra1 (neo-Tra1) were normalised to the bait, Spt7. Ratios from two independent experiments are plotted individually with the mean (black bar). Source data are provided as a Source Data file