The absence of Kif5b impairs MC degranulation in vitro and in vivo. (A) Release of β-hexosaminidase from IgE-sensitized WT and cKO^Kif5b BMMCs, as induced by 20 ng/ml DNP-HSA and determined at the indicated time points. Results are quoted as the mean ± SD of 10 experiments. (B) IgE-sensitized WT and cKO^Kif5b BMMCs were either not stimulated or were stimulated with 20 ng/ml DNP-HSA for 10 min. Cells were fixed, and the cell surface expression of CD63 (as a surrogate marker of degranulation) was determined by flow cytometry (left) and quantified (ΔMFI CD63⁺; right). (C) GFP or GFP-Kif5b was transfected in Kif5b-deficient BMMCs. GFP⁺ BMMCs were then assessed for CD63 expression using flow cytometry (left), and expression was quantified (ΔMFI CD63⁺; right). (D) GFP or GFP-Kif5b DN was transfected in WT BMMCs. GFP⁺ BMMCs were then assessed for CD63 expression using flow cytometry (left), and expression was quantified (ΔMFI CD63⁺; right). (E) WT (n = 5) and cKO^Kif5b (n = 5) mice were passively sensitized with 1 µg/g anti-DNP IgE antibody. 24 h later, mice were challenged with (500 µg) DNP-HSA. Systemic anaphylaxis was determined from the change in body temperature. (F) Serum MCPT-1 levels in WT (n = 5) and cKO^Kif5b (n = 5) mice, measured 60 min after the DNP-HSA challenge. (G, top) IgE-sensitized WT and cKO^Kif5b BMMCs were plated on fibronectin-coated glass coverslips and then stimulated by the addition of 20 ng/ml DNP-HSA for the indicated times. NS, nonstimulated. Cells were then fixed, permeabilized, and stained with anti-STX3. Bars, 2 µm. (Bottom) The percentage of cells with STX3 recruitment to the periphery of the cell. In each individual experiment in the nonstimulated condition (n = 3), stimulated condition for 10 min (n = 3), and stimulated condition for 30 min (n = 3), >100 cells distributed in different areas of the glass coverslip were counted. Statistical analyses were performed using unpaired t tests. *, P < 0.05; **, P < 0.005; ***, P < 0.0001.