(A) FRAP assay measuring effect of ubiquitin overexpression on PTEN import (left panel) and quantification of nuclear and cytoplasmic PTEN accumulation rates (right panel). Error bars are S. D.
(B) Nuclear/ cytoplasmic fractionation of PTEN after ubiquitin overexpression. Lamin B1 and α-Tubulin serve as nuclear and cytoplasmic markers, respectively. Numbers denote nuclear to cytoplasmic PTEN ratios (relative to Lamin and Tubulin, see Supplemental Data). A non PTEN-specific band is indicated (n).
(C) Immunofluorescence imaging of PTEN and K289E with ubiquitin co-expression. Scale bar, 10 μm.
(D) Ubiquitination rescues K289E nuclear import defect as measured by FRAP (left panel; quantifications: right panel). Note that mono-ubiquitination is most efficient in rescuing. Error bars show S. D.