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. 2015 Dec 1;26(24):4412–4426. doi: 10.1091/mbc.E15-02-0087

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© 2015 Li 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 7: — Arf6 is activated in response to NGF stimulation. (A) Analysis of activation of Arf6-GFP after TrkA stimulation. Lysates from PC12 (615) cells expressing Arf6-GFP and stimulated with NGF for the indicated periods of time were incubated with GST or GST-GGA3 PBD, which specifically binds the GTP-bound form of Arf proteins. Bound proteins were analyzed by immunoblotting with anti-GFP antibodies. Bottom, Ponceau staining showing GST-tagged proteins. (B) Comparison of endogenous Arf1, Arf3, and Arf6 activation in response to NGF. Lysates from PC12 (615) cells stimulated with NGF for 15 min were incubated with GST or GST-GGA3 PBD. Bound proteins were analyzed by immunoblotting with anti-Arf1, Arf3, and Arf6 antibodies. The 5% input is shown. Bottom, Ponceau staining showing GST-tagged proteins. (C) Effect of Src, PKC, and PKA inhibitors on NGF-mediated Arf6 activation. The GST-GGA3 PBD pull-down assay were performed as described in A, except that the cells were preincubated for 1 h with specific inhibitors for Src (PP2), PKC (CHE), or PKA (H89) before NGF stimulation for 15 min. Bottom, Ponceau staining showing GST-tagged proteins.