GEF-H1 accumulates at the IS of B cells and is required for exocyst assembly. (A) Representative confocal images of B cells activated with BCR ligand⁺ coated beads for different times. GEF-H1 (green) and α-tubulin (red). Scale bar: 3 µm. (B) B cells were activated on BCR ligand⁺ coated plates for different times, and p-GEF-H1 and total GEF-H1 were detected by Western blot. Representative of three independent experiments. (C) Detection of GEF-H1 in synaptic membranes. B cells were stimulated with BCR ligand⁺ Dynabeads for the indicated times, and GEF-H1 was detected in isolated synaptic membranes by Western blot. Representative of two independent experiments. (D) B cells were activated as described in B, and Exo70 was immunoprecipitated (IP) from corresponding lysates. Associated GEF-H1 and Sec5 were detected by Western blot. Representative of four independent experiments. (E) Control or GEF-H1–silenced B cells were activated as in B, and the formation of exocyst complexes was evaluated by immunoprecipitating Exo70 from lysates. Sec8, Sec5, Sec3, Sec6, and Exo70 were detected by Western blot. Red arrow specifies the band corresponding to Sec5. Representative of two independent experiments.