Figure 5.

Refeeding of the fasted mice leads to Stat5 tyrosine phosphorylation and activation in insulin target tissues. (A) Tyrosine phosphorylation of Stat5 in liver, muscle, and adipose tissues of refed mice. Mice were starved for 3 days, allowed to feed for 80 min, and then sacrificed. Whole cell lysates were prepared from the indicated tissues. Three milligrams each of the total protein lysates was used for immunoprecipitation with anti-Stat5. A second immunoprecipitation from the supernatant was then performed with anti-IR antibody. Anti-Stat5 immunoprecipitates were divided into two parts: one part for PTyr Western blotting (Top; faint signal of Stat5 tyrosine phosphorylation in the heart of refed mice in the original filter did not reproduce well), and the other part for anti-Stat5 blotting (Upper Middle). The complexes captured by anti-IR was assayed in vitro for kinase activity (Lower Middle). The same filter was used for detecting the IR amount by Western blotting with anti-IR antibody (Bottom). (B) Stat5 DNA binding activity is induced by refeeding. Cytoplasmic and nuclear extracts from the livers of fasted and refed mice were subjected to EMSA analysis with the PIE probe (29). Arrow indicates the refeeding-induced complex.