Examination of the correlation between Akt Thr308 and Ser473 phosphorylation and the phosphorylation of Akt substrates. Triplicate samples of normal and patient-matched tumour tissues were separated on SDS–PAGE gels. Phosphorylation of Akt (on Thr308 and Ser473) and three downstream substrate proteins (PRAS40-Thr246, TSC2-Ser939 and TBC1D4-Thr642) was determined by western blotting with phospho-specific antibodies. The data were quantified by densitometry and the strength of evidence of difference in phosphorylation between the normal and tumour samples was determined by Kruskal–Wallis test. Phosphorylation of each protein/site was then coded according to whether it increased (black), decreased (grey) at P⩽0.05 or remained unchanged (white; P>0.05) in the tumour samples relative to normal tissue. The coded data were then grouped according to the direction of change in Thr308 phosphorylation: patients in group I showed an increase, group 2 a decrease and group 3 no change. Group I were further subdivided according to the change in Akt Ser473 phosphorylation (group Ia showing an increase or no change, and group Ib a decrease).