FIG. 5.

Differentiation-dependent Akt and MAP kinase activation is altered in IRS-1 KO cells. The three different cell lines (designated as in Fig. 2) were differentiated as described in Materials and Methods. (A) Lysates were subjected to immunoprecipitation (IP) with a p85-specific antibody followed by immunoblotting (IB) with a pY antibody. Alternatively, lysates were immunoblotted with a phospho-specific Akt (B) or a phospho-specific MAP kinase antibody (C). (D, E) Confluent IRS-1-deficient cells (day 0) were induced in the presence or absence of the MEK inhibitor PD098059 (PD; 50 μM). At day 6 of differentiation, cells were either stained with oil red O (D) or prepared for Western blot analysis of several adipogenic marker proteins as shown (E). A representative experiment of at least two independent experiments is shown.