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. 2010 Oct 4;285(49):38710–38719. doi: 10.1074/jbc.M110.137414

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© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 7. — The binding between the PH domain of CAPS1 and ARF5 induces the accumulation of GDP-locked ARF5 in the Golgi membrane. A–F, subcellular localization of the C-terminal EGFP-HA-tagged CAPS1(PH), the C-terminal FLAG-tagged GDP-locked ARF5(T31N), and endogenous VAMP4 in PC12 cells. A–C, immunostaining of CAPS1(PH)-EGFP-HA and ARF(T31N)-FLAG with anti-HA (A) and anti-FLAG (B) antibodies, respectively, and the merged image (C). D–F, immunostaining of ARF5(T31N)-FLAG and endogenous VAMP4 with anti-FLAG (D) and anti-VAMP4 (E) antibodies, respectively, and the merged image (F). Scale bars (C and F), 10 μm. G–I, subcellular localization of the C-terminal HA-tagged CAPS1(dPH) and the C-terminal FLAG-tagged ARF5(T31N) in PC12 cells. Immunostaining of CAPS1(dPH)-HA and ARF5(T31N)-FLAG with anti-HA (G) and anti-FLAG (H) antibodies, respectively, and the merged image (I). Scale bars, 10 μm. J–M, subcellular localization of endogenous ARF5 in PC12 cells transfected with EGFP together with either control (J and K) or CAPS1 siRNA (L and M). The arrows indicate EGFP-expressing siRNA-transfected cells. Shown is immunostaining of endogenous ARF5 with anti-ARF5 antibody (K and L). Scale bars (K and M), 10 μm. N, ARF5 immunoreactivity levels merged with immunoreactivity for the Golgi complex. PC12 cells were transfected with the Golgi marker B4galt1-tdTomato together with either control (white bar) or CAPS1 siRNA (black bar). The ratio of ARF5 immunoreactivity in the Golgi complex (merged with B4galt1-tdTomato fluorescence) and that in whole cell soma was qualified. **, p < 0.01 by Student's t test. Error bars, S.E. O and P, subcellular distribution of GTP-locked ARF5(Q71L)-FLAG (O) and GDP-locked ARF5(T31N)-FLAG (P) expressed in primary cultured mouse hippocampal neurons. Cultures were transfected at 6 days in vitro, fixed at 8 days in vitro, and immunostained with anti-FLAG antibody. ax, axon; de, dendrite. Scale bar, 50 μm.