FIG. 5.

Effects of Akt-AA on insulin-induced activation of endogenous Akt and MAP kinase in CHO cells. CHO cells cultured in 6-cm-diameter plates were infected with AxCAAkt-AA at the indicated MOI (PFU/cell), incubated for 10 min in the absence or presence of 100 nM insulin, and lysed. (A) Cell lysates were subjected either to immunoblot analysis with polyclonal antibodies to Akt (top) or to three sequential rounds of immunoprecipitation with antibodies to HA in order to deplete Akt-AA, after which the final supernatant was subjected to immunoblot analysis with polyclonal antibodies to Akt (bottom). (B and C) The Akt-AA-depleted final supernatant from panel A was subjected to immunoprecipitation with polyclonal antibodies to Akt (B) or with antibodies to MAP kinase (C), and the precipitates were assayed for Akt kinase or MAP kinase activity, respectively. Data are representative of three independent experiments, with bars representing means of duplicate determinations.