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. 2017 Mar 6;216(3):641–656. doi: 10.1083/jcb.201604025

Figure 3.

Figure 3.

Importin-11 protects PTEN from the NEDD4-1/NDFIP1 lysine ubiquitination system. (A) Western blot and quantification showing that VFP-ΔN–IPO11 regulates PTEN levels through the NEDD4-1– and NDFIP1-mediated ubiquitination pathway (biological replicates [nbiol] = 3). (B, top) Mutation of the PTEN Ub-adduct lysines (PTENK13E, K289E) rescues PTEN protein levels upon VFP-ΔN–IPO11 overexpression in PC3 cells. A black line in the PTEN row indicates splicing because of erroneous inverted loading. (bottom) The PTENK13E, K289E mutant is defective for nuclear import. Bar, 10 µm. nbiol = 4. (C) The t1/2 of GFP-PTENK13E, K289E (red line) is extended compared with that of GFP-PTEN (blue line) as shown by protein stability analysis using cycloheximide: quantification (left) of PTEN Western blots (right). P = 0.01. Error bars are SD of duplicates (see Fig. S2 F). (D) A coIP of IPO11 and GFP-PTEN/GFP-PTENK13E, K289E shows that mutation of PTEN Ub-adduct lysines impairs PTEN–IPO11 interaction in HEK293 cells (nbiol = 2). IB, immunoblot.