Murine and human leukemia cells pre-incubated in DMEM high glucose medium (Thermo Scientific) containing 10 % FBS for 1 hour at 37 °C, then treated for 30 minutes at 37°C using following small molecule inhibitors: dasatinib (LC laboratories, Woburn, MA), saracatinib (Selleckchem, Houston,TX), bosutinib (Selleckchem), p505-15 (Selleckchem), R406 (Selleckchem), R778 (fostamatinib, Selleckchem), RK24466 (Cayman Chemical, Ann Harbor, MI), LCKi-II (Fisher Scientific, Waltham, MA), A770041 (Axon Medchem, Reston,VA), ibrunitinib (PCI-32765, Selleckchem), buparlisib (Selleckchem), or trametinib (LC laboratories).
For intracellular staining, cells were fixed with 1.5% formaldehyde for 10 minutes at room temperature, permeabilized with 100% ice-cold methanol for 20 minutes on ice, and washed twice with staining buffer as described previously (9 (link),10 (link)), and stained with conjugated antibodies to intracellular phospho-proteins (Supplementary Table S2 ). Data were analyzed using Flowjo software.
For intracellular staining, cells were fixed with 1.5% formaldehyde for 10 minutes at room temperature, permeabilized with 100% ice-cold methanol for 20 minutes on ice, and washed twice with staining buffer as described previously (9 (link),10 (link)), and stained with conjugated antibodies to intracellular phospho-proteins (
