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. 2013 Sep 15;24(18):2907–2917. doi: 10.1091/mbc.E13-01-0014

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© 2013 Arasaki 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 6: — RINT-1 modulates TGN SNARE complex assembly. (A) Lysates of cells expressing each of the FLAG-SNARE constructs were immunoprecipitated with anti-FLAG-beads (lanes 7–12) and analyzed by immunoblotting with antibodies against RINT-1 (top) and FLAG (bottom). Five percent input was also analyzed (lanes 1–6). (B) Interaction between RINT-1 and syntaxin 16. Lysates of cells expressing FLAG–syntaxin 16 and each of the GFP–RINT-1 constructs were immunoprecipitated with anti-FLAG beads and analyzed by immunoblotting with antibodies against GFP (middle) and FLAG (bottom). Five percent input was also analyzed by immunoblotting with an anti-GFP antibody (top). (C) SNARE domain is responsible for the interaction with RINT-1. Lysates of cells expressing each of the FLAG–syntaxin 16 constructs were immunoprecipitated with anti-FLAG beads and analyzed by immunoblotting against RINT-1 (middle) and FLAG (bottom). Five percent input was also analyzed by immunoblotting with an ant-RINT-1 antibody (top) (D) TGN SNARE complex assembly is abrogated in RINT-1–depleted cells. HeLa cells were mock transfected (lanes 1 and 3) or transfected with RINT-1 (1149) (lanes 2 and 4). At 72 h after transfection, cell lysates were immunoprecipitated with an anti-Vti1a antibody and analyzed by immunoblotting with the indicated antibodies (lanes 3 and 4). Five percent input was also analyzed (lanes 1 and 2). The intensities of immunostained bands were quantitated with ImageJ. The quantitative data represent the average of two independent experiments.