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. 2014 Dec 17;16(2):232–239. doi: 10.15252/embr.201439260

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© 2014 The Authors. Published under the terms of the CC BY NC ND 4.0 license

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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Direct SH3-SH3 domain-mediated interaction between endophilin A1 and intersectin 1

Endogenous endophilin A1 and intersectin 1 form a complex in situ. Immunoprecipitation from detergent-extracted rat brain synaptosomal fractions (P2′) using control rabbit non-immune IgG (rb IgG) or anti-endophilin A1 antibodies. Samples were analyzed by immunoblotting for endophilin A1 (EndoA1), intersectin 1 (ITSN1), and Hsc70.

GST-intersectin 1-SH3 associates with endophilin A1 present in detergent-lysed rat brain extract (RBE). Samples were analyzed by immunoblotting for endophilin A1 (EndoA1), dynamin 1 (DynI), actin, and GST.

Endophilin A1 associates with intersectin 2. (C) GST or GST-endophilin A1-SH3 fusion proteins were incubated with detergent-lysed rat brain extract (RBE). Samples were analyzed by immunoblotting for intersectin 1 (ITSN1), intersectin 2 (ITSN2; note: antibody specificity was verified by samples from intersectin 2 knockout mice), dynamin 1 (DynI), or actin. (D) Same as in (C) but using detergent extracts from HEK293 cells expressing intersectin 1-eGFP (ITSN1-GFP) or intersectin 2-eGFP (ITSN2-GFP). Samples were analyzed by immunoblotting for eGFP or actin.

Direct binding of endophilin A1-SH3 to intersectin 1-SH3B. Indicated GST-fusion proteins were incubated with full-length (FL) His₆-endophilin A1 (E), His₆-intersectin 1-SH3B (F), or His₆-intersectin 1-SH3A (G). Samples were analyzed by SDS–PAGE and staining with Coomassie blue.