SLAP interacts with multiple Flt3 phosphotyrosine residues independent of the Src interaction sites.
(A) COS-1 cells were co-transfected with pcDNA3-SLAP-FLAG and pcDNA3-Flt3-Y589/591/599F expression vectors. Twenty four hours after transfection, cells were serum-starved overnight and stimulated by FL or not for 5 minutes before lysis. Cell lysates were immunoprecipitated with anti-FLAG antibody followed by Western blotting analysis. (B) Phospho-peptides corresponding to 12 tyrosine phosphorylation sites in Flt3 were immobilized on UltraLink. Peptide-bound slurry was incubated with SLAP-transfected COS-1 cell lysates and pulled-down proteins were then processed for Western blotting using an anti-SLAP antibody.