Constitutive tyrosine phosphorylation of Flt3 and ligand-dependent activation of MAP kinase and Akt.
After a 12-hour starvation period, 1 × 107 cells were stimulated for 10 minutes in 1 mL medium with or without 100 ng/mL FL for 10 minutes as indicated. Immunoprecipitates with anti-Flt3 were performed where indicated, and the immunoprecipitates or total cell lysates were separated by SDS-PAGE. After blotting, the blots were stained with the indicated activation-specific antibodies. pErk recognizes Erk1 (p44), and Erk2 (p42), phosphorylated on tyrosine 204; pAkt is specific for Akt phosphorylated on serine 473. Subsequently, the blots were stripped and stained with the indicated protein-specific antibodies to demonstrate equal loading: tErk is specific for total Erk2 (p42) and does not recognize Erk1.
