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

FIGURE 4.

FIGURE 4.

The PH domain of CAPS1 binds to the N-terminal region of ARF5. A, in vitro binding assay using bacterially expressed recombinant proteins reveals the involvement of the PH domain of CAPS1 in binding to ARF5. MBP-tagged ARF5 protein was pulled down by glutathione-Sepharose beads on which GST, GST-tagged C2 and PH domain (C2 + PH), C2 domain (C2), and PH domain (PH) proteins were immobilized, followed by Western blot analysis (IB) with anti-ARF5 (top) and anti-GST (bottom) antibodies. B, ARF5 binds CAPS1 via the N-terminal region. The region of ARF5 that binds to CAPS1 was screened by generating a series of Ala substitutions in ARF5(T31N)-FLAG at 16 amino acid positions, 3, 4, 6, 11, 17, 62, 101, 108, 109, 137, 146, 152, 162, 164, 176, and 180, all of which are unique to class II ARF4/5 among the ARF family of proteins. CAPS1-HA and Ala-substituted ARF5(T31N)-FLAG constructs were coexpressed in COS-7 cells, and cell lysates were subjected to coimmunoprecipitation (IP) with anti-HA antibody followed by Western blot analysis with anti-FLAG (top) and anti-HA (bottom) antibodies.