Immunohistochemistry. Rabbit anti-AKT pS473 was used at a 1:100 dilution to detect AKT by immunohistochemistry in human breast tumor tissue. The staining is much stronger than the weak basal level of phosphorylation in normal breast. Rabbit anti-AKT pS473 antibody was used with no pretreatment of tissue. Signal was developed using Dako's Techmate streptavidin-biotin reagents. Tissue was formalin-fixed and paraffin embedded.
Immunoblotting. Rabbit anti-AKT pS473 was used at a 1:200 dilution to detect phosphorylated AKT by Western blot. A nuclear extract from cells infected with adenovirus expressing nuclear-targeted AKT kinase was used.