Fig. 2. Coronin 3 binds glucose-induced GDP-Rab27a through the β-propeller structure. (A) Immunoprecipitation of COS-7 extracts that express FLAG-Rab27a mutants and GFP-coronin 3 (upper panel) or GFP-SHD (lower panel) using anti-GFP antibody, and immunoblotting using anti-GFP and anti-FLAG antibodies. 0.2% of the input protein was co-immunoprecipitated. (B) In-vitro-binding assay of purified FLAG-coronin 3 and Rab27a mutants and immunoblotting using anti-FLAG and anti-GST antibodies. (C) Immunoprecipitation of COS-7 extracts that express FLAG-Rab27a-T23N and GFP-coronin 3 deletion mutants using anti-GFP antibody, and immunoblotting using anti-FLAG antibody. 2.0% of the input protein was co-immunoprecipitated except for WT (0.2%). (D) The indicated concentrations of MBP–coronin-3-C were incubated with beads conjugated to GST-Rab27a-T23N; the K_d value was calculated using Scatchard analysis. (E) COS7 lysates that express T7-Rab27a mutants were incubated with beads conjugated to GST-SHD or GST-coronin-3-C. The bound proteins were analyzed by immunoblotting using anti-T7 antibody. (F) MIN6 cells stimulated with 20 mM glucose were extracted and incubated with beads conjugated to GST-SHD or GST-coronin-3-C. The bound proteins were analyzed by immunoblotting using anti-Rab27a antibody.