Activation of the PI3-K/Akt cascade and inactivation of PTEN in freshly isolated CLL cells. Basal levels of main components of PI3-K/Akt cascade in freshly isolated PBMCs of patients with CLL (n = 44) and healthy donors (HDs) were evaluated by Western blotting (the corresponding densitometric scanning is shown in Table 1). A CLL sample (*) was included in Western blotting 2 times together with the HD sets as an internal control. As shown (A), the total amount of PDK1 protein and its phosphorylated form is significantly higher in CLL samples compared with HDs (P < .001 for PKD1 and PDK1-pSer241). As shown (B), the total amounts of Akt and Akt pSer-473 varied between patients with CLL but were higher than in HD samples (P < .05 for Akt pSer-473). The amounts of Akt pThr-308 were significantly higher in most of the CLL samples compared with HD samples (P < .01). As shown (C), comparable amounts of total PTEN were detected in CLL and HD samples. Higher amounts of phosphorylated PTEN at Ser-380, pSer-370, pSTS (pSer-380/pThr-382/pSer-385; P < .01 for all) and CK2 pSer-209 (P < .001) were detected in CLL samples. As demonstrated in panel D, the expression of total PTEN (t) and PTEN pSer-380 in the cytoplasmic (c) and nuclear (n) compartments of purified CLL cells from 3 patients. SOD and ORC2 represent markers for cytoplasmic and nuclear subfraction and show the purity of cellular fractions. Cytoplasmic and nuclear distribution of PTEN-p380 is visualized by immunofluorescence staining in purified CLL cells (E).