Immunoblotting
Immune populations were isolated as previously described and cells were
lysed in 0.5% NP-40, 50 mM Tris-HCl pH 7.4, 5 mM MgCl2and cOmplete™ protease inhibitor cocktail (Roche). Post-nuclear lysates
were resolved by SDS-PAGE using 8% Bolt Bis-Tris Mini Protein Gels
(Invitrogen) and proteins were transferred to Immunoblot PVDF membranes
(Bio-Rad). Membranes were stained with primary antibodies against Flt3
(AF768, R&D systems), phosphorylated tyrosine (4G10, Sigma-Aldrich),
actin (A5060, Sigma-Aldrich) or tubulin (DM1A, Abacm) followed by
HRP-conjugated secondary antibodies.