Figure 3: Conformational change of PTEN monitored by BRET following mutation of key regulatory residues.

(a) Schematic representation of PTEN showing the regulatory phosphorylation site residues in the C-terminal tail and the mutation introduced to create the PTEN-A4 mutant. Respective mBRET (b), ΔmBRET (c) and %ΔmBRET (d) values of Rluc-PTENwt-YFP and Rluc-PTEN(A4)-YFP. The graphs represent mean±s.e.m. of four independent experiments. ***P<0.001, t-test. (e) Localization of PTEN constructs in HEK cells. Cells were transfected with Rluc-PTENwt-YFP and Rluc-PTEN(A4)-YFP, and live cells were imaged. Insets show magnified regions of the boxed areas and demonstrate membrane targeting of PTEN-A4 (arrowheads). (f) % ΔmBRET of Rluc-PTEN(A4)-YFP versus Rluc-PTENwt-YFP in HeLa cells. Error bars are s.e.m. (g,h) Colocalization of Rluc-PTEN-YFP and Rluc-PTEN(A4)-YFP with fluorescent-wheat germ agglutinin (WGA) on non-permebilized HeLa cells. Insets show magnified regions of the boxed regions. Scale bars, 10 μm. Quantification mean±s.e.m. on 12 different cells was performed using the Image J colocalization plugin, ***P<0.001, t-test. BRET measurements in these experiments were performed on detached cells.