Subcellular localization of the kinesin-1–dependent transport machinery in MCs. (A–D) Confocal microscopy of WT or Kif5b-deficient BMMCs transfected with GFP-Rab27b and Dsred-Slp3 and labeled with anti-STX3 antibody in resting cells (A and C) or in cells stimulated by the addition of 20 ng/ml DNP-HSA for 10 min (B and D). (E) The statistical analysis of experiments in B and D was performed using an unpaired t test. ***, P < 0.0001. More than 30 cells were counted per setting. (F–J) IgE-sensitized WT (F and H) and cKO^Kif5b (G, I, and J) BMMCs were plated on fibronectin-coated glass coverslips and either were not stimulated (F, G, and I) or were stimulated by the addition of 20 ng/ml DNP-HSA for 10 min (H and J). Cells were then fixed, permeabilized, and stained with an anti-tubulin antibody and an anti-Kif5b or anti-STX3 antibody. Bars, 2 µm. All images of single cells are representative of >100 cells observed over at least three independent experiments. (K) Statistical analysis of microtubule reorganization at the plasma membrane identified by filopodia-like extension shape in experiments in H and J was performed using an unpaired t test. More than 100 cells were counted per setting. NS, not stimulated.