Sucrose Density Gradient Separation of IgE-Sensitized BMMCs

Sucrose density gradient separations were performed as previously described (42 (link)). Briefly, IgE-sensitized BMMCs isolated from WT or ORMDL2,3 dKO mice were used. BMMCs (15 × 10⁶) were then activated or not with antigen (TNP-BSA; 0.25 μg/ml) for 5 min. After centrifugation the cells were lysed on ice in 0.8 ml lysis buffer (10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 2 mM EDTA, 10 mM glycerophosphate, 1 mM Na₃VO₄, 1 mM PMSF, protease inhibitor cocktail) supplemented with 1% Brij-96. The gradient was formed by adding 0.5 ml of 80% sucrose stock solution to the bottom of the polyallomer tube (13 × 51 mm; Beckman Instruments) followed by 1.5 ml of 40% sucrose containing the cell lysate, 2 ml of 30% sucrose, and 1 ml of 10% sucrose. After 4 h centrifugation at 4°C and 210,000g, using an SW55 Ti rotor, nine fractions were collected from the top of the gradient, and individual fractions were size-fractionated by SDS-PAGE followed by immunoblotting with PY20-HRP conjugate or LYN-specific antibody. The position of phosphorylated PAG, LYN, LAT1, and LAT2 is known from our previous studies (43 (link), 44 (link)).