Effect of MgcRacGAP on M1 cell sensitivity to IL-6–induced differentiation. MgcRacGAP renders M1 cells more sensitive to the IL-6–induced differentiation signal. (A) Quantitation of GFP expression in the M1 cells at 4 days after retroviral gene transduction on flow cytometry in pMX-IG, pMX-IG/FL, or pMX-IG/STAT3F. The x-axis indicates fluorescence intensity as a log scale ranging from 10⁰ to 10⁴. The y-axis indicates the number of the cells. Parental M1 cells were used as a control. (B) Expression of the Flag-tagged FL and HA-tagged STAT3F in M1 transfectants. Cell lysates from parental M1, M1/pMX-IG, and M1/pMX-IG/FL cells (1 × 10⁷ per lane) were immunoprecipitated and examined by means of immunoblotting and an anti-Flag M2 monoclonal antibody (i). Cell lysates from parental M1, M1/pMX-IG, and M1/pMX-IG/FL cells (1 × 10⁷ per lane) were immunoprecipitated with the use of the anti-HA monoclonal antibody (12CA5) and immunoblotted with anti-HA rabbit polyclonal Ab (ii). (C) Quantitation of cell differentiation on flow cytometry in unstimulated parental M1 cells and M1 cells expressing pMX-IG, pMX-IG/FL, or pMX-IG/STAT3F at 4 days after IL-6 treatment (5 ng/mL). Differentiated M1 cells were detected in region R2. (D) M1 cells expressing pMX-IG, pMX-IG/FL, or pMX-IG/STAT3F were incubated for 4 days with various concentrations of IL-6. The cells were then analyzed for the differentiation on flow cytometry. The percentages of differentiated cells were evaluated by the percentages of the cells in the R2 region. The result shown is representative of 3 experiments.