Coexpression of SOCS1 and FLT3-ITD. (A) Simultaneous equimolar coexpression of SOCS1 and FLT3-ITD does not affect FLT3-ITD–mediated signaling pathways. Western blot analysis of retrovirally transduced stable 32D cells expressing FLT3-ITD or SOCS1-T2A-FLT3-ITD or control (empty vector). Membranes were incubated with indicated phospho-specific antibodies and were reprobed for the nonphospho–specific-antibodies and β-actin to ensure equal loading. Two independent bulk cultures (1 and 2) of SOCS1-T2A-FLT3-ITD were used to exclude effects clonality. (B) Proliferation assay by measurement of DNA synthesis using [³H]thymidine incorporation. The stable 32D cells expressing the indicated constructs were analyzed for their proliferation by [³H]thymidine incorporation as described under “Methods.” Each data point represents the mean of [³H]thymidine incorporation of quadruplicates, with SD as error bars. (C) 32D cells stably expressing either empty vector control, SOCS1-T2A-FLT3-ITD, or FLT3-ITD were stimulated with either IFN-α (1000 U/mL) and IFN-γ (100 ng/mL) for 10 minutes or left unstimulated. Lysates were resolved by SDS-PAGE and immunoblotted for phospho-STAT1 and SOCS1, as indicated. β-actin served as a loading control. (D-E) 32D cells transduced with control, SOCS1, FLT3-ITD, and SOCS1-T2A-FLT3-ITD retroviruses were monitored for GFP-positivity (percentage) every 24 hours by FACS and cultured without IL-3 (D) or with IL-3 (E). Time is plotted on the x-axis, with the percentage of GFP positivity from 3 independent experiments, with SD as error bars, plotted on y-axis (*P < .05).