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. 2016 Jan 18;212(2):167–180. doi: 10.1083/jcb.201506130

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© 2016 Niño et al.

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Figure 6. — Nup60 ubiquitylation participates in the Rad53-mediated pathway. (A) 6His-Ub-conjugated forms of Nup60-HA were extracted from asynchronous cells (AS) or from cells treated with α-factor for 3 h before release for indicated periods of time and analyzed as in Fig. 1. The bottom panel shows DNA content analysis by flow cytometry. (B) His-Ub protein purified from cells collected 60 min after release from α-factor arrest were treated (+) or not (−) with AP, and Nup60-HA ubiquitylation was analyzed by Western blotting. (C) rad53K227A cells expressing 6His-Ub were synchronized with α-factor for 3 h before release for indicated periods of time and analyzed as in Fig. 1. (D) Serial dilutions of WT and mutant cells were spotted on YPD without or with HU or MMS at the indicated concentrations and grown at 30°C.