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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 2. Purification and identification of the PTEN ubiquitin ligase as NEDD4-1.

Figure 2

(A) The purification scheme. The detailed procedure was described in Experimental Procedures.

(B) The PTEN E3 activity in fractions (Frac, 2 μl each) from the final Mono Q column was measured by the in vitro PTEN ubiquitination assay.

(C) The final Mono Q fractions (15 μl each) were resolved by SDS-PAGE and stained with silver.

(D) The purified PTEN E3 activity can ubiquitinate HA-tagged recombinant PTEN. The purified E3 (2 μl) or HS100 (5 μl) was used in the reactions as indicated. The ubiquitinated PTEN was detected by immunoblotting against HA-tag.

(E) The domain structure of NEDD4-1. Amino acid positions of individual domains are denoted by corresponding numbers.

(F) Interaction of NEDD4-1 with PTEN in 293T cells determined by coimmunoprecipitation analysis. PTEN and HA-tagged NEDD4-1 (N4-HA) were transfected as indicated. After immunoprecipitation using anti-HA antibody, PTEN and N4 in the precipitates were detected by immunoblotting (the upper two panels). The cell lysate (before immunoprecipitation) was blotted for N4, PTEN, and tubulin (the lower three panels) as controls.

(G) Direct physical interaction of PTEN with NEDD4-1 (N4) determined by a GST pull-down assay. GST-PTEN but not GST alone pulled down purified NEDD4-1.