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. Author manuscript; available in PMC: 2018 Nov 20.
Published in final edited form as: Curr Biol. 2017 Nov 2;27(22):3454–3467.e8. doi: 10.1016/j.cub.2017.10.014

Figure 1. PER degradation is finely tuned by reversible ubiquitination.

Figure 1

(A) PER1 and 2 have the shortest half-lives among essential clock proteins. Per2Luc MEFs were treated with CHX and harvested at the indicated times. The arrow indicates a nonspecific band in the PER1 blot. These results were replicated in three independent experiments. (B) PER is robustly ubiquitinated when cells are treated with the DUB inhibitor, b-AP15, but not other DUB inhibitors. The PER1 blot was stripped for the PER2 blot. The arrow indicates a nonspecific band. (C) The drugs induced dramatically elevated ubiquitination in total proteins. Note that treatment with b-AP15 or PR-619 almost depleted free ubiquitin (Ubi). Two different Ubi blots are shown. (D) Overexpressed PER proteins are also rapidly ubiquitinated. PER and CRY were overexpressed more than 20-fold above endogenous counterparts in MEFs. (E) The slow migrating PER species are polyubiquitinated PERs. (F) PER2 is ubiquitinated when cotransfected with 6xHis-Ubi. PER2 and 6xHis-Ubi were coexpressed in HEK293a cells. See also Figures S1 and S2A-C.