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. 2016 Jan 11;14(1):e1002347. doi: 10.1371/journal.pbio.1002347

Fig 6. OTUB1 N22 hydroxylation does not impact on OTUB1 protein level and half-life.

Fig 6

(A) HEK293 cells stably expressing shOTUB1 (targeting the 3′UTR to diminish endogenous OTUB1 protein) and FLAG-OTUB1 WT or N22A were transiently transfected with FIH-V5 for 24 h followed by western blot analysis of OTUB1 WT and N22A protein levels at the indicated time points. (B) Densitometric analysis of OTUB1 WT and N22A protein levels. OTUB1 protein levels were normalized to β-actin loading control. (C) HEK293 wild-type cells were transiently transfect with either control plasmid or pFIH-V5 (to maximize OTUB1 hydroxylation) for 24 h and subsequently treated with either 1 mM DMOG (to prevent OTUB1 hydroxylation) or DMSO for the indicated time points. Endogenous OTUB1 protein levels were analyzed by western blot. (D) Densitometric analysis of endogenous OTUB1 protein levels. OTUB1 protein levels were normalized to β-actin loading control. (E) HEK293 wild-type cells were pre-treated with DMSO or 1 mM DMOG (to inhibit OTUB1 hydroxylation) for 16 h prior to the treatment with 50–100 μg/ml cycloheximide (CHX) for the indicated time points. Endogenous OTUB1 protein levels were analyzed by western blot. (F) Densitometric analysis of endogenous OTUB1 protein levels. OTUB1 protein levels were normalized to β-actin loading control. Data are presented as representative blot or as mean ± SEM of n = 3 independent experiments. The underlying data of panels B, D, and F can be found in S1 Data.