To demonstrate the phosphorylation of STAT 1 [pS727], 3T3- L1 cells were treated with 50 nM insulin for 15 minutes, followed by treatment with 100 units/mL IFN-gamma for 15 minutes. Proteins from serum-starved (Lane 1) and serumstarved/ insulin and INF gamma treated (Lane 2) 3T3-L1 cell extracts were resolved by SDS-PAGE on a 4-20% Trisglycine gel. The proteins were then transferred to nitrocellulose. Membranes were incubated with 1 µg/mL anti-STAT 1 [pS727]. After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStar detection method. The Western blotting data indicate that STAT 1 is phosphorylated at serine 727 upon exposure to insulin followed by IFN-gamma.