Figure 9: Trastuzumab induces conformational changes that correlate with enhanced lipid phosphatase activity of PTEN.

(a) BRET experiment in SKBR3 and MCF-7 cells transfected with Rluc-PTEN-YFP and treated with trastuzumab (2 μg ml⁻¹) for the indicated times. Graph represents means±s.e.m. for four independent experiments. Lower panel shows representative western blotting using an anti-ErbB2 antibody on lysates of SKBR3 and MCF-7 cells. GAPDH was used as loading control. (b) In vitro phosphatase assay of immunoprecipitated Rluc-PTEN-YFP from transfected SKBR3 treated with Trastuzumab for 20 min (2 μg ml⁻¹). YFP vector control values were subtracted and samples normalized to PTEN alone. Graph represents the mean±s.e.m. of three independent experiments ***P<0.001, t-test. (c) Levels of activated Akt (pAkt) assessed in SKBR3 cells. Cells were transfected with Rluc-PTEN-YFP and treated with Trastuzumab (2 μg ml⁻¹). a.u., arbitrary units. Graph represents the mean±s.e.m. of four independent experiments *P<0.05, t-test. (d) MCF-7 cells stably transfected with ErbB2 or the truncated ErbB2 p95 variant were transiently transfected with Rluc-PTEN-YFP and treated with trastuzumab (2 μg ml⁻¹) for the indicated times. Graph represents means±s.e.m. for four independent experiments, *P<0.05, t-test. Lower panel shows representative western blotting using an anti-ErbB2 antibody on lysates. Tubulin was used as loading control. BRET measurements in these experiments were performed on adherent cells.