Fig. 4. Constitutive membrane localisation of the isolated PH domain. (A) HEK293T cells were transfected with expression vectors for Gab1-GFP or the isolated PH domain fused to GFP together with vectors encoding EpoR-gp130 chimeric receptors. 24 hours after transfection, cells were transferred to poly-L-lysine-coated coverslips. The next day, cells were starved for 3 hours in KRH and transferred into the perfusion chamber of a laser-scanning microscope. After 20 minutes, the cells were stimulated with Epo (7 U/ml). Gab1 localisation is shown before stimulation and after 20 minutes of Epo stimulation. (B) HEK293T cells were transfected with expression vectors for Gab1-GFP wild type or the mutants Gab1-PH-GFP, Gab1-PI3K-GFP, Gab1-Grb2-GFP or Gab1-SHP2-GFP, together with vectors encoding EpoR-gp130 chimeric receptors. Cells were stimulated and Gab1 localisation analysed as described in A.