Autophosphorylation of FLT3 in leukemia blast cells shows a time-dependent pattern after conditioned medium is replaced with fresh medium. (A) Frozen leukemia blast cells were thawed, washed, and cultured in fresh medium for 12 hours. Cells (4 × 106 cells/mL) were then washed twice and incubated in fresh medium. Cells were collected at 0, 3, 5, and 7 hours (lanes 2-5) and lysed. Another 10 × 106 cells were lysed right after being thawed and washed (lane 1). 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). (B) Frozen leukemia blast cells were thawed, washed, and cultured in fresh medium for 12 hours. Cells were then washed twice and incubated with fresh medium with or without IMC-NC7 antibody (10 μg/mL) at a density of 4 × 106 cells/mL. Cells were collected at 0, 0.5, 1, 3, and 5 hours and lysed in protein lysis buffer. Lysates were then analyzed as in panel A.
