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. 2012 Sep 1;23(17):3370–3379. doi: 10.1091/mbc.E11-12-1010

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© 2012 Wasik 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: — Septin 7 is a novel interaction partner of CD2AP and nephrin. (A) GST-fused 3.SH3 domain of CD2AP or GST alone were incubated with rat glomerular lysate, and the bound proteins were separated by SDS–PAGE, which was followed by staining with GelCode blue. The <50-kDa band detected in the CD2AP-GST pulldown was identified as septin 7 by mass spectrometry. (B) Septin 7 is pulled down with GST-2.SH3 and 3.SH3 domains of CD2AP, but not with GST alone, from rat glomerular lysate. (C) Schematic structure of septin 7 and GST-septin 7 fusion protein constructs used for pulldown assays. PB, polybasic region; CC, coiled-coil domain; FL, full-length (aa 1–418); N, NH₂ terminus (aa 1–31); Center, central region (aa 32–298); C, COOH terminus (aa 179–418), and C2, COOH terminus 2 (aa 244–418). (D) Pulldown assays with GST-septin 7 fusion proteins covering full-length septin 7 (septin 7-FL), septin 7-center, septin 7 NH₂ terminus (septin 7-N), and septin 7 COOH-terminal fragments (septin 7-C2 and septin 7-C) show that full-length septin 7 and the COOH terminus of septin 7 (septin-7-C) pull down CD2AP from rat glomerular lysate. (E) Two different CD2AP antibodies (CD2AP 211 and 209) coimmunoprecipitate endogenous septin 7 in HEK293 cells. Immunoprecipitation with septin 7 antibodies indicates septin 7 band immediately below the IgG heavy chain. Neither septin 7 nor CD2AP are present in immunoprecipitations with normal rabbit serum (NRS). (F) GST-nephrin cytoplasmic domain, but not GST alone, pulls down septin 7 from rat glomerular lysate. (G) Pulldown assays with GST-septin 7 fusion proteins indicate that the central domain of septin 7, but not GST alone, pulls down nephrin from rat glomerular lysate. (H) Nephrin coimmunoprecipitates with septin 7 antibodies, but not with rabbit IgG from rat glomerular lysate. Isolated glomeruli or HEK293 cells were lysed in 1% NP-40, 20 mM HEPES (pH 7.5), and 150 mM NaCl, incubated with CD2AP, septin 7, or nephrin GST-fusion proteins or antibodies, and the precipitated proteins were immunoblotted with anti-CD2AP, anti–septin 7, or anti-nephrin IgG. Glomerular or HEK293 cell lysates (10 μg) are included as control.