Immunoprecipitation
cDCs from wild type (+/+), heterozygote (+/ITD) or homozygote (ITD/ITD) littermates were isolated and stimulated in 100 ng/mL Flt3L in complete RPMI at 37ºC and 10% CO2 for 0, 10 or 30 minutes. Cells were washed and lysed in 0.5% NP-40, 50 mM Tris-HCl pH 7.4, 5 mM MgCl2 and cOmplete™ protease inhibitor cocktail (Roche). The lysate was pre-cleared with protein G-sepharose beads (WEHI Antibody Facility) and normal rat and mouse serum (WEHI Antibody Facility). Flt3 was precipitated with anti-Flt3 (AF768, R&D systems) antibody and protein-G sepharose beads. Proteins were eluted with reducing SDS sample buffer and analyzed by SDS-PAGE and immunoblotting.