TSC2 and neoplasm: Three separate samples of tumour and three samples of patient-matched normal tissue were lysed and subjected to SDS–PAGE followed by western blotting for the level of phosphorylation of Akt on Thr308 and Ser473, as well as the phosphorylation of the well-characterised downstream Akt substrates PRAS40 (on Thr246), TSC2 (on Ser939) and TBC1D4 (on Thr642).