Incubation of leukemic blast cells in complete medium for 12 hours increases the FLT3 autophosphorylation in some primary AML specimens. Protein lysates were prepared from washed, newly thawed AML blast cells with or without FL (100 ng/mL) stimulation or after incubation for 12 hours in complete medium. Total cellular protein was immunoprecipitated with anti-FLT3 antibody. The immunoprecipitates were resolved by 8% SDS-PAGE and subjected to immunoblot analysis with antiphosphotyrosine antibody 4G10 (top panel). The same blot was stripped and reprobed with anti-FLT3 antibody (bottom panel).