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. 2016 Jul 1;27(13):2008–2013. doi: 10.1091/mbc.E16-03-0180

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© 2016 Tanabe 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 1: — Construction of cTAGE5 point mutants lacking Sec12-binding ability. (A) 293T cells were transfected with FLAG tag only, FLAG-tagged cTAGE5 coil1 (aa 61–300), cTAGE5 coil2 (aa 301–650), cTAGE5 (aa 61–221), or cTAGE5 (aa 115–300) with HA-tagged Sec12 (aa 1–386). Cell lysates were immunoprecipitated with anti-FLAG antibody and eluted with the FLAG peptide. Eluates and cell lysates were analyzed by SDS–PAGE followed by Western blotting with FLAG or HA antibodies. (B) Alignment of human cTAGE5 (aa 61–115) with corresponding regions from other species. Identical amino acids are shaded in black, and similar amino acids are shaded in gray. (C) 293T cells were transfected with FLAG tag only, FLAG-tagged cTAGE5 wild type, cTAGE5 (aa 61–300) T1, or cTAGE5 (aa 61–115) T1 with HA-tagged Sec12 or TANGO1. (D) 293T cells were transfected with FLAG tag only, FLAG-tagged cTAGE5 wild type, cTAGE5 R61A S65A, S68A R69A, Y71A, E75A K76A, K89A L93A, S97A, or L112A with HA-tagged Sec12. (E) 293T cells were transfected with FLAG tag only, FLAG-tagged cTAGE5 wild type, E75A K76A, K89A L93A, or S97A with HA-tagged TANGO1. (F) 293T cells were transfected with FLAG tag only, FLAG-tagged cTAGE5 wild type, K89A L93A, or K89A with HA-tagged Sec12 or TANGO1. (C–F) Cell lysates were immunoprecipitated with anti-FLAG antibody and eluted with the FLAG peptide. Eluates and cell lysates were analyzed by SDS–PAGE followed by Western blotting with FLAG or HA antibodies.