Fig 1: Diminished phosphorylation response of signaling effectors in FceRI-stimulated BMMCs and IgG stimulated neutrophils and monocytes in the context of IRAP-deficiency. (A–C) WT and IRAP-deficient (IRAPKO) BMMCs were sensitized with anti-DNP IgE for 24 hours before stimulating them with specific antigen (DNP-HSA). After indicated time points, stimulation was arrested by adding fixation and permeabilization buffer. Phosflow analysis of signaling effectors (left panel) was assessed using anti-pSykY519/520 (A), anti-pLATY200 (B) and anti-pp38Y182 (C). Total levels of proteins in non-stimulated cells were also analyzed using anti-Syk, anti-LAT and anti p38 (right panels). Phosphorylation levels were determined as the ratio of the gMFI of samples divided by the gMFI of the FMO of the respective sample and represent the mean ± s.e.m of indicated experiments. Statistical analysis was done using a Student’s test. (D, E) Phosflow analysis of Syk phosphorylation at Y519/520 was also determined on ex vivo IgG-stimulated neutrophils and monocytes during ASA. Cells were accessed directly from the blood 5 min after initiation of ASA. The gating strategies for analysis of BMMCs, neutrophils and monocytes and representative examples of Ab staining are shown, respectively, in ( Supplementary Figures 2B, C ). Data shown are analyzed as in (A–C) and presented as the mean ± s.e.m of indicated experiments. Statistical analysis was done using a Student’s test. *: P < 0.05; **: P < 0.01; *** P < 0.001. ns, not significant.