PKB/Akt is expressed but not constitutively phosphorylated in B-CLL.

B-CLL cells were lysed immediately after separation from peripheral blood. (A) Whole protein lysates (100 μg) from 7 different patients were analyzed by immunoblotting with the antibodies as indicated. NIH-3T3 cells treated with platelet-derived growth factor (PDGF) served as a positive control (pc); without PDGF as a negative control (nc). (B) Akt in vitro kinase assay was performed after immunoprecipitation of Akt from 7 different patients; GSK-3 fusion protein served as exogenous substrate for Akt. Kinase reaction was analyzed by immunoblotting with mAb specific for phospho-GSK-3 (Ser21/9). (C) To rule out in vitro phosphorylation of Akt, B-CLL cells were incubated with or without 10 μM LY294002 and lysed after 12 and 24 hours.