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. 2022 Dec 5;54(12):1919–1932. doi: 10.1038/s41588-022-01223-8

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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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Extended Data Fig. 2 — a. Schematics for endogenously tagging CTCF, RAD21, WAPL with the mAID degron and for endogenously tagging YY1 with miniIAA7. b. Immunoblots of CTCF, RAD21, WAPL, YY1, and their tags (HaloTag for CTCF, V5 for RAD21, RFP (mScarletI) for YY1, and HA for WAPL) for the protein expression levels and sizes in wild type mESCs and degron clones C58 (ΔCTCF), F1 (ΔRAD21), YD39 (ΔYY1), and C40 (ΔWAPL). c. Quantification of the levels of WAPL, CTCF, Rad21 and YY1 proteins in the degron clones C58 (ΔCTCF), F1 (ΔRAD21), C40 (ΔWAPL) and YD39 (ΔYY1) relative to wild type mESCs by immunoblotting (n = 2 independent immunoblots ran on the same cell lysates). Black asterisks point to the basal degradation level of each degron-tagged factor in the corresponding cell line. d. Immunoblots of CTCF, RAD21 and WAPL proteins across a degradation time course from 0 (untreated) to 3 hr (IAA treatment) in ΔCTCF, ΔRAD21, and ΔWAPL degron clones. e. Schematic for biochemical salt fractionation experiment in mock-treated (UT) or IAA-treated degron clones. f. Immunoblots of cytoplasmic (Cyt) and nuclear proteins dissociating from chromatin at increasing salt concentrations (75, 150, 300 and 500 mM NaCl) as schematized in g, probed with the indicated antibodies (α). Son: sonicated, solubilized chromatin; % of total: signal intensity of each fraction divided by the total signal intensity across all fractions; % of degradation: 1 – (signal intensity of each fraction in the IAA treated condition divided by the untreated condition), after normalization for total protein amounts (normalizer for ΔWAPL degron: total CTCF; normalizer for ΔRAD21 degron: total YY1). A blot with anti-histone 2B antibody (almost exclusively found in the solubilized chromatin) controls for chromatin integrity during the fractionation steps.

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