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. 2011 Jul 29;6(7):e22209. doi: 10.1371/journal.pone.0022209

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Wang 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) Plasmids carrying RAD6 or rad6 mutants were transformed into the strains UCC6389 with RAD6 deletion expressing HTB1 or htb1-T122E. WCEs prepared from the indicated strains were analyzed by western blot. H2B and its ubiquitylation were detected by anti-Flag antibody; H3 K4 and K79 trimethylation were analyzed using anti-H3 K4me3 or anti-H3 K79me3 antibodies. The G6PDH antibody was used to monitor protein loading. (B) Overnight cultures of the indicated strains were 5-fold serial diluted and spotted on the indicated plates (-His-Lys for plasmid selection; -His-Lys+FOA for telomere silencing analysis). (C) H2Bub1 does not affect chromatin condensation in htb1-T122E cells, as shown by sucrose gradient centrifugation. Vector only or plasmids with WT RAD6 were transformed into rad6Δ htb1-T122E yeast with UCC6389 background. The relative positions of the fragments containing sub-telomeric (sub-TEL), or euchromatin region (euC), were shown in upper panel or lower panel, respectively.