CD34+ peripheral blood progenitors from human blood and bone marrow aspirates from patients with SM were obtained following informed consent under protocols approved by the NIAID Institutional Review Board (98-I-0027 and 02-I-0277). The characteristics of these patients are specified in Table S1 in Supplementary Material. Primary HuMC cultures were derived from CD34+ progenitors as described (32 (link), 33 (link)); and mononuclear cells from marrow aspirates were separated in a Ficoll gradient and cultured for 5 days in StemPro media supplemented with 100 ng/mL SCF (34 (link)).
HMC-1.1 and HMC-1.2 were kindly provided by Dr. Butterfield at the Mayo Clinic. HMC-1 cells, LAD-2 cells, and murine bone marrow-derived mast cells (BMMCs) from Sphk1-, Sphk2-deficient, and WT mice were cultured as described (28 (link), 35 (link), 36 (link)). P815 mastocytoma murine cells with a mutation homologous to the human D816V mutation were from ATCC (Manassas, VA, USA). HCT116 cells with or without the D816V-KIT introduced by CRISPR were from Thermo Fisher Scientific. P815 and HCT116 cells were cultured as specified by the respective providers. Cell lysates for Western blots were prepared from 1 × 106 cells and processed as described (37 (link)).
HMC-1.1 and HMC-1.2 were kindly provided by Dr. Butterfield at the Mayo Clinic. HMC-1 cells, LAD-2 cells, and murine bone marrow-derived mast cells (BMMCs) from Sphk1-, Sphk2-deficient, and WT mice were cultured as described (28 (link), 35 (link), 36 (link)). P815 mastocytoma murine cells with a mutation homologous to the human D816V mutation were from ATCC (Manassas, VA, USA). HCT116 cells with or without the D816V-KIT introduced by CRISPR were from Thermo Fisher Scientific. P815 and HCT116 cells were cultured as specified by the respective providers. Cell lysates for Western blots were prepared from 1 × 106 cells and processed as described (37 (link)).
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