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. 2011 Jun 1;22(11):1943–1954. doi: 10.1091/mbc.E10-11-0923

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© 2011 He et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 2: — Netrin-1–activated Akt mediates interaction between PIKE-A and UNC5B. (A) Netrin-1 increases the binding of PIKE-A to UNC5B. Wild-type and GTPase-dead (KS) PIKE-A were cotransfected into HEK293 cells with HA-UNC5B. The transfected cells were treated with netrin-1 for 30 min. PIKE-A was immunoprecipitated with anti-Myc antibody and analyzed with anti-HA antibody. Equal amounts of HA-UNC5B and Myc-PIKE-A were immunoprecipitated (second and third panels). (B) PIKE-A interaction with UNC5B in LN-Z308 cells. LN-Z308 cells were treated with netrin-1 for 30 min, endogenous UNC5B was immunoprecipitated with control IgG or anti-UNC5B antibody and the bound PIKE-A was detected using anti–PIKE-A antibody (top). Confirmation of Akt and PIKE-A phosphorylation status (fourth and fifth panels). (C) The UNC5B death domain is essential for the interaction between UNC5B and PIKE-A. The diagram of UNC5B domains is shown (top). Different UNC5B truncates were cotransfected into HEK293 cells with mGST–PIKE-A. PIKE-A was pulled down with glutathione beads and analyzed with anti-HA antibody. Truncation of the death domain in UNC5B or its whole intracellular domain abolished the interaction between PIKE-A and UNC5B (bottom). (D) PI 3-kinase signaling regulates the interaction between PIKE-A and UNC5B. HA-UNC5B and Myc–PIKE-A cotransfected HEK293 cells were pretreated with PD98059 (10 μM), wortmannin (100 nM), LY294002 (10 μM) for 30 min, before netrin-1 was introduced. Myc–PIKE-A was immunoprecipitated using Myc antibody, and the bound HA-UNC5B was detected using anti–HA-HRP antibody. Netrin-1 triggered the interaction between PIKE-A and UNC5B, but PI 3-kinase inhibitor markedly blocked it (top). Confirmation of Akt phosphorylation status (bottom). (E) Netrin-triggered PIKE-A phosphorylation is Akt dependent. LN-Z308 cells were infected with ad-shRNA-Akt for 36 h followed by 200 ng/ml netrin-1 treatment for 30 min. Immunoblotting was conducted with anti–phospho-PIKE-A Ser-472 (top), anti-Akt (middle), and anti–PIKE-A (bottom) antibodies.