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. 2011 Oct 1;22(19):3684–3698. doi: 10.1091/mbc.E11-04-0308

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© 2011 Fu 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: — Recruitment of endophilin A1 to APPL1 endosomes requires retrolinkin activity. (A) Hippocampal neurons transfected with the retrolinkin shRNA construct were starved for 2 h, treated with BDNF (25 ng/ml), fixed, and immunostained with antibodies to pTrk, endophilin A1, and RFP. Quantification of colocalization is shown (right). Data represent means ± SEM (n = 15) (**p < 0.01). (B) Hippocampal neurons transfected with the endophilin A1 shRNA construct were stimulated with BDNF and immunostained with antibodies to pTrk, retrolinkin, and RFP. (C) Quantification of colocalization between pTrk and endophilin A1 or retrolinkin, respectively. Data represent means ± SEM (n = 15). Scale bar, 10 μm. (D) CoIP of HEK293 cells overexpressing EGFP-APPL1 and FLAG-TrkB or FLAG-endophilin A1. Cell lysates were incubated with immobilized FLAG antibody. Immunoblot was probed with antibodies to FLAG and GFP. (E) Hippocampal neurons transfected with control or retrolinkin shRNA construct were immunostained with antibodies to endophilin A1, APPL1, and RFP after BDNF treatment. (F) Quantification of the colocalization between endophilin A1 and APPL1. Data represent means ± SEM (***p < 0.001; n = 15). (G) Hippocampal neurons transfected with the control or endophilin A1 shRNA construct were immunostained with antibodies to retrolinkin, APPL1, and RFP after BDNF treatment. Scale bar, 10 μm. (H) Quantification of the colocalization between retrolinkin and APPL1. Data represent means ± SEM (n = 15). EEN1, endophilin A1; RTLN, retrolinkin.