Fig. 6. Rapid tyrosine phosphorylation of membrane-associated STAT5 after treatment with EGF. (A) Cytosol and membrane fractions were prepared from treated and untreated SKBR3 cells, as described in the legend to Fig. 4. Samples were subjected to SDS-PAGE and immunoblotting with anti-STAT5 antibodies. (B) Immunoblotting evidence for the co-precipitation of STAT5 with EGFR and, to a lessor extent, to ErbB2/ErbB3. Treatment of cells is indicated. (C) Immunoblotting with anti-STAT5 PY694 antibodies demonstrates the time course of STAT5 phosphorylation in response to 20 nM EGF. Membrane sheets in D-F were prepared from serum starved SKBR3 cells (D) or after 2 minutes of treatment with 20 nM EGF (E,F). Sheets in E and D were singly labeled with 5-nm gold reagents that specifically recognize STAT5 when phosphorylated on Tyr694; there is marked increase in the amount of phospho-STAT5 (encircled) but not in the general pool of STAT5 after EGF treatment. The membrane sheet in F was double labeled for phospho-STAT5 (5-nm gold) and EGFR (10-nm gold). Boxed areas mark the few co-clusters of EGFR and phospho-STAT5 in EGF-treated membranes. Bars, 0.1 µm.