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. Author manuscript; available in PMC: 2008 Jan 12.
Published in final edited form as: Cell. 2007 Jan 12;128(1):129–139. doi: 10.1016/j.cell.2006.11.039

Figure 1. Polyubiquitination of PTEN in vivo and in vitro.

Figure 1

(A) Polyubiquitination of transfected PTEN. C-terminal His-tagged PTEN was co-transfected with HA-tagged ubiquitin (HA-Ub) in 293T cells as indicated. The cells were treated with or without 25 μM proteasome inhibitor MG132 for 4 hours and then harvested and lysed. His-tagged PTEN was pulled down with Ni2+ beads, washed with 6 M guanidine as described in Experimental Procedures, and subjected to immunoblotting against HA-tag to detect ubiquitinated PTEN.

(B) In vitro ubiquitination of PTEN. The in vitro assay was performed as described in Experimental Procedures, with individual components added as indicated. GST-PTEN was used as the substrate, and HeLa S-100 (HS100, 5 μl and 5 mg protein/ml) was required to provide the PTEN ubiquitin ligase (E3) activity.