Autophosphorylation of FLT3 in EOL-1 cells shows a time-dependent pattern after conditioned medium is replaced with fresh medium. EOL-1 cells (4 × 10⁶ cells/mL) were washed twice and then cultured in fresh medium with or without IMC-NC7 antibody (10 μg/mL). Cells were collected at 0, 1, 3, 5, 7, and 9 hours and lysed in protein lysis buffer. 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).