Choline kinase regulates Akt activity. A, MDA-MB 468 cells were transfected with 50 nM of the indicated pool siRNA. 3 days posttransfection, 50 μM LY294002 was added to scr transfected cells for 30 mins. Whole cell lysate were subjected to western blotting with the indicated antibodies. B, siRNA transfection and western blot were performed as (A). Percentage of pAkt(ser473) signal were calculated by quantifying the pAkt(ser473) normalized to total Akt on the immunoblot and compared to the scr control (set as 100%). C, MDA-MB 231 cells were transfected with 50 nM of indicated pool siRNA for 2 days. Cells were serum-starved overnight and stimulated with IGF for 15 mins. Whole cell lysates were harvested and western blot performed with the indicated antibodies. The values below the blot indicate the ratio of normalized pAkt(ser473) signals quantified using Image J to that of scr control (set as 1). D, MCF7 were transfected with 1 μg pcDNA vector, ChoK A or B plasmids for 24 h using Lipofectamine 2000 (Invitrogen). Cells were harvested and ChoK activity determined as described in the methods. E, MCF7 was transfected as in (D) and western blot with indicated antibodies.