Surface expression and autophosphorylation of Flt3 receptors. 32D cells were transfected with constructs encoding either Flt3-WT or Flt3-D835Y or Flt3-ITD. (A) Flow cytometric analyses of Flt3 surface expression on 32D cells. The shaded curves show staining with anti-Flt3 antibody; the open curves, the isotype control. (B) Flt3 was immunoprecipitated from 32D cells expressing the indicated Flt3 constructs. Immunoprecipitates were resolved on 4% to 15% SDS-PAGE and immunoblotted for 4G10, an antibody specific for phosphotyrosine. Equal loading was confirmed by reprobing with an antibody recognizing activation independent state of Flt3.