For some experiments, thighbones from Endocrinology antagonist Lyn−/− and Lyn+/+ mice 18 were kindly provided by Dr. Toshiaki Kawakami (La Jolla Institute of Allergy and Immunology). C57BL/6J mice were purchased from Charles River Laboratories Japan (Kanagawa, Japan). Following the approval of a committee of Nihon University, all experiments were performed in accordance with the guidelines for the care and use of laboratory animals of Nihon
University. Cultures of BMMC were prepared from the femurs of 4- to 8-wk-old mice as previously described 19. For retroviral transfection, BM cells were cultured in the presence of 100 ng/mL recombinant SCF for another 7 days. The ecotropic retrovirus packaging cell line PLAT-E, https://www.selleckchem.com/products/LDE225(NVP-LDE225).html which was kind gift from Dr. Toshio Kitamura (Tokyo University., Japan), was maintained in DMEM supplemented with 10% v/v FBS, 1 μg/mL puromycin
(BD Clontech, San Jose, CA, USA) and 10 μg/mL blasticidin S (Kaken Pharmaceutical, Tokyo, Japan). Retroviral gene transduction into FcRβ−/− mast cells was performed as previously described 20. Briefly, pMX-puro plasmids harboring WT (αβYYYγ2) or mutated (αβFFFγ2, αβFYFγ2, and αβYFYγ2) FcRβ cDNA were transfected into PLAT-E to generate recombinant retroviruses. BM cells were infected with the retroviruses for 48 h in the presence of 10 μg/mL polybrene (Sigma). The gene-transduced cells were selected with 1.2 μg/mL puromycin for 7 days. Viable cells (10–20% of the BM cells cultured with retroviruses) were expanded for several weeks. Puromycin-resistant transfectants, which express cell surface FcεRI at comparable levels, were used for experiments. Degranulation was determined by β-hexosaminidase release as described previously 19. The percentage of net β-hexosaminidase release was calculated as follows: (supernatant optical density of the stimulated cells – supernatant optical density
of the unstimulated cells)×100/(the total cell lysates optical density of unstimulated cells – supernatant optical density value of the unstimulated cells). For up-regulation of FcεRI expression isometheptene at the cell surface, mast cells (1×106/mL) were incubated with 0.5 μg/mL of IgE for 4 or 48 h. The cells were stained with 0.1 μg/mL of anti-mouse IgE mAb conjugated with FITC at 4°C for 30 min. The stained cells were analyzed with FACSCalibur (BD Biosciences). Stimulated mast cells (1×106) were washed twice with ice-cold PBS and lysed for 30 min on ice in lysis buffer (Tris-buffered saline containing 1% Nonidet P-40, 2 mM PMSF, 10 μg/mL aprotinin, 2 μg/mL leupeptin and pepstatin A, 50 mM NaF and 1 mM sodium orthovanadate). The lysates were centrifuged for 15 min at 15 000 g. For immunoprecipitation, the cells (1–3×107) were lysed in lysis buffer containing 0.25% Triton-X100 instead of 1% Nonidet P-40. The cell lysates were incubated with antibody bound-Protein G Sepharose for 3 h on ice. The immunoprecipitates were resuspended in an equal volume of 2× Laemmli buffer.