Enhancement of MgcRacGAP-STAT3 interaction by IL-6 stimulation. (A) Lysates from IL-6–stimulated M1 cells (50 ng/mL for the time indicated) were incubated with a similar amount of MBP-Myo, MBP-Cys, or MBP-GAP bound to beads. Bound proteins were separated on SDS-PAGE and immunoblotted with anti-STAT3 Ab or anti-Rac1 Ab. (B) MgcRacGAP partially colocalized with STAT3 at the cytoplasm and at the nucleus in M1 cells. With the use of unstimulated (i-iv) and IL-6–stimulated (v-viii) M1 cells, immunostaining for MgcRacGAP (i,v) and STAT3 (ii,vi) was done. For the merge figures (iv,viii), small insets on the right show the field at a high magnification to demonstrate the detail of the colocalization of MgcRacGAP and STAT3. The immunostained coverslips were viewed with a fluorescence microscope IX70 (Olympus). The scale bar indicates 2 μm or 0.6 μm. (C) Direct interaction of MgcRacGAP with STAT3-DNA–binding domain in vitro. Full-length MgcRacGAP was expressed in Sf9 cells with the use of the baculovirus vector and was purified from infected Sf9 cells. The recombinant MgcRacGAP was pulled down by MBP-STAT3-DBD– or MBP-bound beads, then subjected to Western blot analysis with anti-MgcRacGAP (i) or anti-MBP Ab for the loading control (ii).