Fig. 6. In vivo immunoprecipitation using anti-Akt1 antibodies. Primary mouse hepatocytes were stimulated with insulin for 30 min, and total cell lysates were prepared. Akt1 and Foxa-2 proteins were identified in cell lysates by immunoblotting. Akt1 was then precipitated by using polyclonal anti-Akt-1 antibodies (Cell Signaling) bound to gammabind-sepahose (Amersham Pharmacia) for 16 h at 4° C. Control cell lysates (lane 3) were treated identically but in the absence of anti-Akt-1 antibodies. Proteins were eluted with SDS-loading buffer, separated by 12% SDS/PAGE, and analyzed by Western blotting, using either anti-Akt1 or anti-Foxa-2 antibodies and secondary antibodies linked to horseradish peroxidase (Calbiochem). Proteins were visualized by chemiluminescence detection using the ECL system (NEN).