Influence of the PH domain and the activation status of Akt.

<p><b>A</b> Domain structure of Akt <b>B</b> Purified GST-tagged active Akt or a mutant lacking the PH domain were incubated with purified FLAG-tagged FKBP51. After 3 h, the interaction was tested by GST pulldown and Western blotting. <b>C</b> and <b>D</b> HEK293T cells were transfected with the indicated HA-tagged Akt1 phosphorylation site mutants with or without co-transfected FLAG-tagged FKBP51^K352A/R356A (TPR_mut). After 2 days cells were collected, lysed, immunoprecipitated and analyzed by Western blotting (duplicates were analyzed for D). <b>E</b> HEK293T cells were starved for 16 h, stimulated with FCS for 45 min or treated with wortmannin. Controls were incubated in the presence of 10% FCS and treated with DMSO. Cells were lysed, immunoprecipitated and analyzed by Western blotting.