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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 3.

Figure 3

In vitro ubiquitylation assay of n-PTEN and 4p-PTEN by NEDD4-1 and XIAP. (A) The reaction for n-PTEN and 4p-PTEN ubiquitylation by NEDD4-1 was conducted with 5 mM ATP, 100 μM wild-type ubiquitin, 50 nM E1, 1 μM E2 (UbcH5c), 1 μM NEDD4-1, and 10 μM PTEN, for 0, 0.5, 1, 2, and 4 h. The samples were analyzed by colloidal blue staining. (B) n-PTEN and 4p-PTEN ubiquitylation (1 μM) by NEDD4-1 under the same reaction conditions was analyzed by Western blotting using the anti-PTEN antibody. (C) The ubiquitylation of n-PTEN and 4p-PTEN by XIAP was analyzed by colloidal blue staining. The reaction of the mixture containing 100 μM wild-type ubiquitin, 50 nM E1, 1 μM E2 (UbcH5c), 1 μM XIAP and 1 μM PTEN was quenched at 0, 1, 2, and 4 h and analyzed by colloidal blue staining. (D) Ubiquitylation of n-PTEN and 4p-PTEN under the same condition as in panel C was analyzed by Western blotting using the anti-PTEN antibody.