Figure 1: The Rluc-PTEN-YFP biosensor retains PTEN functionality.

(a) Schematic representation of Rluc-PTEN-YFP biosensor. PTEN is sandwiched between Rluc (Luc in diagram) and YFP. This diagram illustrates how conformational changes may induce variations in BRET measurements, although the real orientations of donor and acceptor proteins are not known. (b) PTEN immunoprecipitates from PC-3 cells expressing YFP or Rluc-PTEN-YFP were analysed for the capacity to dephosphorylate water-soluble diC8-PIP3 (100 μM). Data shown represent mean±s.e.m. of three independent experiments. **P<0.01, t-test. (c) Levels of activated Akt (pAkt Ser 473) measured in PC-3 cells recomplemented with Myc-PTEN, Rluc-PTEN-YFP or PTEN-YFP. Histogram represents quantification of relative pAkt levels for four independent experiments. A.U., arbitrary units. ***P<0.001, analysis of variance with Bonferroni post hoc test. (d) Subcellular distribution of YFP, PTEN-YFP and Rluc-PTEN-YFP. HEK cells were transfected with the indicated plasmids and live cells were imaged. Scale bar, 10 μm. (e) Co-immunoprecipitation of Rluc-PTEN-YFP with Myc-β-arr2 from transfected HEK293 cells. Myc-β-arr2 was immunoprecipitated using anti-myc antibodies and associated Rluc-PTEN-YFP was revealed with an anti-green fluorescent protein antibody.