SNX16 physically interacts with Nwk. (A) Schematic of Nwk and SNX16 domain organization and summary of yeast two-hybrid interactions. (B) Alignment of SNX16 homologues between aa 346 and 363 of Drosophila SNX16. NCBI Protein database accession numbers and a full SNX16 alignment are shown in Fig. S1. Shaded glutamate residues are required for Nwk–Snx16 interactions. (C) GST and GST-SNX16 (aa 335–407; coiled coil [CC]) were used to precipitate binding partners from wild-type Drosophila head extracts. Equal amounts of precipitates were immunoblotted with α-Nwk antibodies. The first lane represents 2.5% of the input compared with experimental samples. (D) GST and GST-Nwk (aa 1–731) were used to precipitate binding partners from wild-type, UAS-Snx16, and UAS-Snx16-GFP–expressing Drosophila head extracts. Equal amounts of precipitates were immunoblotted with α-GFP, α-SNX16, or α-Dap160 antibodies. Dap160 has previously been shown to interact with Nwk (O’Connor-Giles et al., 2008; Rodal et al., 2008). The first and fourth lanes represent 2% of the input compared with experimental samples. (E) GST-Nwk (aa 1–731) was coexpressed in E. coli with empty vector, SNX16 (aa 220–407; PX and coiled-coil domains), or SNX16^3A (aa 220–407), precipitated on glutathione agarose, and immunoblotted with α-SNX16 antibodies. A. gambiae, Anopheles gambiae; D. rerio, Danio rerio; R. norvegicus, Rattus norvegicus.