Colocalizer pro pro#

Cells were then stimulated with 100 ng/ml FL for 30 minutes before lysis and immunoprecipitated with an anti-Flt3 antibody. (B) Ba/F3-Flt3 cells with SLAP shRNA or control shRNA were serum-starved and preincubated with cycloheximide for 4 hours. Cells were lysed and lysates were immunoprecipitated with an anti-Flt3 antibody followed by Western blotting analysis. (A) Ba/F3-Flt3 cells with SLAP shRNA or control shRNA were serum-starved and stimulated with 100 ng/ml FL for 5 minutes. SLAP depletion decreases ubiquitination and degradation of Flt3 receptor. C were quantified using multi-gauge software. (D) Signal intensities from three independent experiments of Fig. A were quantified using Multi-Gauge software.(C) Cell lysates were immunoprecipitated with respective antibodies and analyzed by Western blotting. (B) Signal intensities from three independent experiments of Fig. Membranes were then stripped and reprobed with respective antibodies to show the loading. (A) Total cell lysates were separated by SDS-PAGE and membranes were probed with phospho-specific antibodies.

Colocalizer pro pro#

(E) Co-localization of SLAP and Flt3 was determined using CoLocalizer Pro 2.7.1.Įffects of SLAP on FL-induced Akt, Erk and p38 activation.īa/F3-Flt3 cells with SLAP shRNA or control shRNA were treated with 100 ng/ml FL or not for 5 minutes before lysis. Blue: DAPI, green: SLAP-FLAG and red: Flt3. (D) Ba/F3 cells transfected with Flt3-WT and SLAP-FLAG were stimulated with FL for 5 minutes and then stained with fluorophore-conjugated primary antibodies. (C) Ba/F3 cells transfected with Flt3-WT and SLAP-FLAG were stained with fluorophore-conjugated primary antibodies. (B) Lysates of stably transfected Ba/F3-Flt3-WT cells with SLAP shRNA or control shRNA were immunoprecipitated with an anti-SLAP antibody and analyzed by Western blotting. Gray area indicates cells labeled with isotype-matched control and black area indicates cells labeled with Flt3 antibody. (A) Stably transfected Ba/F3 cells were labeled with isotyp-matched control or PE-conjugated Flt3 antibody and analyzed by flow cytometry. 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.Įxpression of SLAP and Flt3 in Ba/F3 cells. (B) Phospho-peptides corresponding to 12 tyrosine phosphorylation sites in Flt3 were immobilized on UltraLink. Cell lysates were immunoprecipitated with anti-FLAG antibody followed by Western blotting analysis. Twenty four hours after transfection, cells were serum-starved overnight and stimulated by FL or not for 5 minutes before lysis.

(A) COS-1 cells were co-transfected with pcDNA3-SLAP-FLAG and pcDNA3-Flt3-Y589/591/599F expression vectors. SLAP interacts with multiple Flt3 phosphotyrosine residues independent of the Src interaction sites. (D) Structure of SLAP showing different domains and known tyrosine phosphorylation sites. Total Flt3 and SLAP phosphorylation was detected by 4G10 and the membrane was further probed with SLAP and Flt3 antibodies. Cells were then processed for immunoprecipitation with an anti-FLAG antibody and Western blotting analysis. (C) SLAP-FLAG and Flt3-WT transfected COS-1 cells were stimulated for different time points after overnight starvation. Cells were then processed for immunoprecipitation and Western blotting analysis. (B) SLAP-FLAG and Flt3-ITD transfected COS-1 cells were stimulated for 5 minutes after overnight starvation. Cell lysates were immunoprecipitated with anti-Flt3 or anti-FLAG antibody followed by Western blotting analysis. (A) COS-1 cells were co-transfected with pcDNA3-SLAP-FLAG and pcDNA3-Flt3-WT expression plasmids. SLAP interacts with Flt3 in response to FL-stimulation.