RAC1 and STAT5 activity are involved in ROS production. (A) CEP-701–mediated inhibition of FLT3/ITD leads to a small decrease in RAC1 activity (RAC1-GTP level). RAC1 was used as a loading control. Indicated at the bottom is the fold change of RAC1-GTP relative to total RAC1. (B) Immunoprecipitation of gp91^phox and Western blotting for RAC1 show decreased binding of RAC1 to gp91^phox after CEP-701 treatment. gp91^phox was used as a loading control. Indicated at the bottom is the fold change of RAC1 relative to total gp91^phox. (C,D) CEP-701 blocks phosphorylation of FLT3, which in turn prevents STAT5 phosphorylation. Western blotting for pFLT3 (C) and pSTAT5 (D) in CEP-701–treated and untreated cells 32D/ITD cells. FLT3 and STAT5 were used as loading controls, respectively. (E,F) Partial knockdown of STAT5, which was confirmed by Western blotting, leads to significant decrease in ROS production in 32D/ITD cells. No significant decrease in ROS was observed in cells transfected with scrambled siRNA (si-Scramble) in comparison with the control without siRNA (CTRL). (G) RAC1-GTP pull-down assay in 32D and 32D/ITD cells, followed by Western blotting for RAC1 and pSTAT5. RAC1-GTP appears increased in 32D/ITD compared with 32D cells. pSTAT5 binds active RAC1 in 32D/ITD cells. Ten percent input protein is shown detected by Western blotting for RAC1.