Fig 1: Absence of IRAP does not affect early signaling events mediated by Lyn kinase. (A) IgE-sensitized WT and IRAP-deficient (IRAPKO) BMMCs were either not stimulated or stimulated with antigen (30 ng/mL of DNP-HSA) before being lysed and immunoprecipitated with anti-FceRIß chain mAb. Levels of co-immunoprecipitated phospho-ß chain (**) and phospho-? chain (*) in non-reduced gels were determined using anti-phospho-tyrosine (PY20) Ab. Total immunoprecipitated FceRI receptors were determined after stripping and reblotting with anti-FceRß chain mAb. A representative blot and quantitative analysis of indicated number of experiments are shown in the left and right panels, respectively. Statistical analysis was done using a Student’s t-test. (B) WT and IRAP-deficient (IRAPKO) BMMCs were sensitized with anti-DNP IgE and were then plated on fibronectin-coated glass coverslips. Cells were stimulated with 30 ng/mL of DNP-HSA for indicated time points. After fixation and permeabilization cells were stained with DAPI (blue), anti-IgE (green) and anti-Lyn (red) Abs as indicated. Cells were analyzed by confocal microscopy. Images show representative sections (out of 2 experiments) with multiple cells for IgE and Lyn single staining as well as the merge of all colors. ns, not significant.