Stat1 in VSMCs is activated in coculture. (A) Stat1 immunofluorescence in VSMCs in coculture with monocytes and monoculture control. Original magnification × 400. (B) VSMCs after coculture were separated by MACS separating system and analyzed for pSer727 Stat1 and pTyr701 Stat1 in whole-cell lysate, cytosolic (c), and nuclear (n) fractions by subjection to SDS-PAGE and Western blotting with anti-pSer727 Stat1 and anti-pTyr701 Stat1 rabbit polyclonal antibodies, respectively. As a control for Stat1 amount, blots were analyzed for Stat1 using anti-Stat1 monoclonal antibody (bottom panel). (C) Gamma-activated site/interferon-stimulated response element (GAS-ISRE)-binding activity of nuclear extracts prepared from VSMCs cocultured with monocytes and from monoculture were analyzed by EMSA. Open arrow indicates the position of the protein-³²P-GAS/ISRE complex, and the solid arrow indicates the position of the free probe.