FIG. 4.

PKBβ is the predominant isoform in adipocytes. (A) Cell lysates (100 μg) from serum-starved 3T3-L1 fibroblasts (Fib) or 3T3-L1 adipocytes (Ad) were depleted of PKBβ by two consecutive rounds of immunoprecipitation with the sheep PKBβ antibody. Twenty micrograms of lysate (Lysate) and one-fifth of the immunoprecipitation supernatant (After PKBβ-IP) were analyzed by SDS-PAGE and immunoblotting with the sheep PKBα or the sheep PKBβ antibodies. (B) Cell lysates (100 μg) from isolated rat adipocytes treated with 1 μM insulin (I) for 15 min or left basal (B) were depleted of PKBβ and analyzed as described for panel A. (C) Immunoprecipitation with the sheep PKBβ antibody was performed on 100 μg of cell lysates prepared from 3T3-L1 adipocytes overexpressing HA-PKBα. The immunoprecipitate (PKBβ-IP) and 10 μg of lysate (Lysate) were analyzed by SDS-PAGE and immunoblotting for the presence of HA-PKBα, with a monoclonal HA antibody.