Constitutively active Akt and MEK2 activate downstream signaling pathways after nucleofection in primary CLL B cells. (A) Immunoblotting analysis of phospho-Akt, phosphoGSK3α/β, phospho-ERK, and HA-tagged MEK2 in CLL B cells collected 48 hours after transfection with empty pcDNA3 vector (Control), myr.Akt, or CA MEK2. Total Akt and total ERK served as loading controls. One representative CLL sample (G159) is shown. (B) Analysis of cell cycle–regulatory proteins 48 hours after nucleofection with empty pcDNA3 vector (Control), myr.Akt, CA MEK2, or cotransfection of Akt and MEK2. The Burkitt lymphoma cell-line BJAB was used as a control for the expression of the various proteins. Actin served as a loading control. The percentage of viable cells was determined by Annexin V/PI staining and is indicated at the bottom of the panel. (C) Flow cytometric analysis shows an increase in leukemic cell size 48 hours after nucleofection with myr.Akt.