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. Author manuscript; available in PMC: 2016 Nov 9.
Published in final edited form as: Biochemistry. 2016 Jun 22;55(26):3658–3666. doi: 10.1021/acs.biochem.6b00448

Figure 6.

Figure 6

In vitro ubiquitylation assay of n-PTEN with wild-type (wt-Ub) or lysine-free ubiquitin (K0-Ub). (A) The ubiquitylation reaction was conducted in 40 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 5 mM ATP, 50 nM E1, 1 μM E2, 1 μM WWP2, 10 μM n-PTEN, with 100 μM wt-Ub or K0-Ub. The reactions were quenched at 0, 0.5, 2, 4, and 6 h and the mixtures analyzed by colloidal blue staining, and the reaction with K0-Ub at 6 h was subjected to mass spectrometry analysis. (B) Same in vitro ubiquitylation conditions as in panel A using wt-Ub or K0-Ub with NEDD4-1.