Phosphorylation of Akt at Thr308 and Ser473 in human breast tumors. IHC analysis of invasive ductal carcinomas for pAkt at Thr308 (A,B) and Ser473 (C-F). (A) Thr308-phosphorylated Akt is both cytosolic and nuclear in distribution. (B) The phospho-Akt (Thr308)-specific blocking peptide inhibits binding of the anti-phospho-Akt (Thr308) mouse monoclonal antibody. Panels C,E show the distribution of Ser473-phosphorylated Akt in tumor cells. (C) Phosphorylated Akt has a partly cytosolic distribution with a predominant staining at the inner cell membrane. Strongly increased Akt phosphorylation at Ser473 in another tumor (E). (D,F) Immunostaining for Akt Ser473 is blocked after pre-incubation with a phospho-Akt (Ser473)-specific blocking peptide. The IHC results are in agreement with the published literature that phosphorylated Akt can be detected in the cytoplasm, and that Ser473 phosphorylation occurs at the inner cell membrane and precedes Thr308 phosphorylation, which leads, as a second step, to membrane detachment and nuclear translocation of phosphorylated Akt [36–38]. Magnification: 100× for E,F; 200× for A,B,C,D. Counterstain: Methyl Green.