BCR engagement triggers the recruitment of Exo70 and Sec3 to the IS and exocyst assembly. (A) Left: Representative confocal images of B cells incubated with nonactivating (BCR ligand⁻) or activating (BCR ligand⁺) beads for 1 h at 37°C. Cells were fixed and stained for endogenous Exo70 (green) and LAMP-1 (red) or transfected with Sec3-HA and stained for HA (green) and LAMP-1 (red). White circles and dashed lines indicate bead position and cell boundaries, respectively. Blue arrowheads on inset point to LAMP-1⁺Sec3⁺ spots at the IS. Scale bar: 3 µm. Right: Quantification of Exo70 or Sec3 polarity indexes toward the IS. Data are shown as box-and-whisker plots. The ends of whiskers represent the 10th and 90th percentile. Unpaired t test; ****, P < 0.0001; Exo70 polarity index was calculated from n ≥ 39 cells from three independent experiments; Sec3, from n ≥ 26 cells from two independent experiments. (B) B cells were stimulated with BCR ligand⁺ Dynabeads for the indicated times, and synaptic membranes were isolated containing BCR-associated protein complexes, followed by Exo70, Sec3, LAMP-1, and GAPDH detection by Western blot. Representative of two independent experiments. (C) Exo70 immunoprecipitation (IP) assay to detect the formation of the exocyst complex in B cells activated for indicated times. Representative of four independent experiments.