Fig 4. Protein detection of AKT2 and AKT2-13a.

HEK293T cells were transfected with either pcDNA3 vectors containing cDNA coding for AKT2 and AKT2-13a or with pEBG-2T vectors which encode AKT2-GST fusion proteins. Cells were lysed after 24 hours and protein extracts were separated by SDS-PAGE followed by blotting and antibody detection with either a AKT2-specific antibody (A) or with an antibody generated to recognize the novel peptide sequence unique for AKT2-13a (B). The AKT2-specific antibody produced double bands at the height of AKT2, the lower of which likely represent degradation products. (C). AKT2 (“WT”) and AKT2-13a (“13a”) were transfected in HEK293 cells as detailed in Materials and Methods. Cells were harvested and protein extracts were prepared. Proteins were separated by SDS-PAGE. Immunoblotting was performed with detection of AKT2 or AKT2-13a and three antibodies specifically detecting the indicated phosphorylated residues (p-T309 is the activation loop; p-452 is in the TM; p-474 is in the HM). (D) HEK293T cells were either transfected with pcDNA-AKT2-13a (left, one dish) or left untransfected (right; thirty dishes). AKT2-13a was immunoprecipitated from protein extracts from these cells using the antibody directed against the unique C-terminal peptide of AKT2-13a. Washed precipitates were analyzed by SDS-PAGE and western blotting using AKT2 antibody detection.