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. 2006 Sep 18;26(22):8267–8280. doi: 10.1128/MCB.00201-06

FIG. 7.

FIG. 7.

Akt2 and Akt1 cytonuclear localization and binding of endogenous p21. A. Cytoplasmic and nuclear fractions from unstimulated (−IGF-I) and IGF-I-stimulated (+IGF-I; 50 nM during 10 min) C2 cells were probed for Akt1 (upper panel) and Akt2 (middle panel) to monitor subcellular distribution by Western blotting using specific isoform antibodies. The same fractions were also probed using anti-tubulin (lower panel) to assess for fractionation efficiency. B. Coimmunoprecipitation experiments from total mouse cell extracts immunoprecipitated (IP) with a polyclonal p21 antibody and blotted with Akt1 (upper panel) or Akt2 (middle panel) using isoform-specific antibodies. The same membranes were subsequently blotted with monoclonal anti-p21 antibody, to show the amount of immunoprecipitated p21. C. Similar experiments performed with primary human fibroblasts and monoclonal anti-p21. Intracellular distribution for Akt1 (upper lanes) and Akt2 (lower lanes) in total cell extracts (Tot) and cytoplasmic (Cyto) or nuclear (Nucl) fractions. Right lanes, p21 immunoprecipitates from nuclear or cytoplasmic fractions probed for the presence of Akt isoforms. D. Immunoprecipitation of p21 from control-, siAkt1-, or siAkt2-treated cells. Shown are Western blot analyses of the immunoprecipitates for Akt2 and p21 protein.