The rat basophilic leukemia RBL-2H3 cells were cultured in monolayers as described [17]. The B2 subclone derived from a Syk-negative variant of RBL-2H3 cells and cells obtained by stable transfection of the B2 with the wild type (WT) or a kinase inactive form of Syk [16] have been kindly provided by Drs. J. Zhang and R. P. Siraganian (National

Institutes of Health, Bethesda, MA, USA). Bone marrow cells were obtained by flushing the femur and tibia bones of C57BL/6 mice and cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 1% X63Ag8-653-conditioned medium as source of IL-3 [38], 2 mM L-glutamine check details and 50 units/mL penicillin. Cells were passed twice weekly at a concentration of 1 × 106 cells/mL, and after 4–6 weeks of culture they were BMMCs as assessed for IgE binding by FACS analysis. Adherent RBL cells were incubated with 0.5 μg/mL of anti-DNP mouse IgE for 12 h at 37°C. BMMCs were incubated with anti-DNP IgE (0.5 μg/106 cells) in RPMI supplemented with 10% FCS for 1 h at 4°C on a rotating wheel. After washing, cells were resuspended at 107cells/mL in medium supplememented with 5% FCS, and stimulated with 1 μg/mL DNP-HSA for the indicated lengths of time. Cell lysis, immunoprecipitation, electrophoresis,

and immuno-blotting were performed as previously described [11, 17]. Particulate membrane and cytosol fractions were isolated from RBL-2H3 cells as previously described [39, 40] Selleckchem Tamoxifen with the following modifications. Upon stimulation cells Axenfeld syndrome were washed with ice-cold PBS, resuspended (5 × 106 cells/mL) in sonication buffer (20 mM HEPES, 10 mM NaF, 1 mM MgCl2, 1 mM EDTA, 1 mM DTT, 5 mM N-ethyl-maleimide, 1 mM Na3VO4) and sonicated 3 × 10 s. Lysates were then centrifuged at 600 × g for 5 min at 4°C and transferred in

polypropylene tubes for ultracentrifugation at 36,000 rpm for 1 h at 4°C using a swing SW60 rotor (Beckman Instruments Inc., Palo Alto, CA, USA). Supernatant was collected as cytosolic fraction while the membrane pellet was resuspended in sonication buffer and protein solubilized by adding a final concentration of 0.5% (v/v) NP-40 detergent. Upon 30 min of centrifugation at 14,000 rpm at 4°C, equal protein amounts of postnuclear supernatants were separated as total cell lysates or immunoprecipitated with anti-Hrs Ab and subjected to SDS-PAGE and immunoblotting. The purity of membrane and cytosolic fractions was verified by probing with anti-FcεRIβ chain and anti-β tubulin mAbs, respectively. Syk-siRNA (5′-CGAGAGAGAUGUACGAC-3′), Cbl-siRNA (5′-GUGAAGAAGACACGGAAUA-3′) and a control nontargeting siRNA (5′-UAAGGCUAUGAAGAGAUACUUTT-3′) were purchased from Eurofins MWG Operon (Ebersberg, Germany). Specific protein knockdown was achieved by transfecting RBL-2H3 cells with siRNA duplexes. The transfection was performed by electroporation (310 V, 960 μF) incubating 1 × 107 cells with 2.5 μM siRNA in 500 μL of serum-free MEM.