Fig. 2. Inhibition of PIP₃-dependent Akt activation by the various PH domains. COS-7 cells were transfected with a HA-tagged full-length Akt construct with or without a construct encoding a plasma-membrane-targeted PI 3-kinase (PI3K-CAAX) and the indicated PH-GFP constructs. One day after transfection and after 6-8 hours of serum deprivation, cells were lysed and the activity of HA-Akt was measured after immunoprecipitation as detailed under Materials and Methods. Equal expression of HA-Akt, as well as the other expressed proteins, was ensured by keeping the total transfected DNA equal by complementing with the respective empty plasmid DNAs during transfection and were determined from the total cell lysates. In each experiment, the Akt activities were normalized to the level observed with PI3K-CAAX in the presence of GFP alone, which gave an average of tenfold increase over the basal. Mean±s.e.m. of three similar observations are shown.