Figure 3.

Stat5b-Ct and endogenous Stat5b are tyrosine phosphorylated in response to insulin stimulation in vivo. (A) Tyrosine phosphorylation of Stat5b-Ct in COS cells. Five micrograms each of pRcCMV-Stat5b-Ct and pECE-IR were cotransfected into COS1 cells. After 48 h, the cells were starved for 2 h in serum-free medium and then stimulated with either insulin (50 nM) or IGF1 (20 ng/ml) for 15 min. HA-Stat5b-Ct was immunoprecipitated with 12CA5 antibody, and the immunoprecipitate was divided into two aliquots, one for Western blotting with RC20 (Left) and the other with horseradish peroxidase-12CA5 (Right). Arrows indicate the positions of Stat5b-Ct. (B) Tyrosine phosphorylation of Stat3 and Stat5 in CHO-IR cells. CHO-IR cells were starved in serum-free medium for 4 h in the presence of 200 mM Na₃VO₄ and then stimulated with insulin (50 nM) for 15 min. One microgram of protein each from the cell lysates was immunoprecipitated with anti-Stat1, Stat3, or Stat5 antibodies. Each immunoprecipitate was divided into two equal parts for detection of Stat tyrosine phosphorylation (Upper) and Stat protein (Lower). (C) Insulin activates Stat3 and Stat5 DNA binding in CHO-IR cells. CHO-IR cells were treated as in B, and nuclear extracts were prepared. Nuclear extracts (20 μg) were subjected to electrophoretic mobility-shift assay (EMSA) analysis with the m67 SIE (Left) or β-casein PIE (Right) probes. Anti-Stat1 and Stat3 antibodies and an antibody against the N-terminal portion of Stat5b (Stat5b-nt) (recognizes both murine Stat5a and Stat5b) were used for the supershift assays. (D) Insulin activates Stat1, Stat3, Stat5a, and Stat5b DNA-binding in HIR3.5 cells. Cells were starved in DMEM with 0.1% BSA for 4 h and then treated with 200 μM vanadate for 40 min, when buffer or insulin (50 nM) was added for the last 15 min. Whole cell lysates were prepared and subjected to gel-mobility shift analysis with the M67 SIE (Left) and β-casein PIE (Right) probes. Antibodies against Stat1, Stat3, Stat5a-Ct, and Stat5b-Ct were used for supershift. Arrows indicate the specific insulin-inducible complexes; asterisks indicate supershifted Stat-containing complexes.