Figure 1

DFsn is identified as a Highwire-interacting protein. (a) The Highwire-associated complex was purified by the TAP procedure (see Materials and methods) and analyzed by one-dimensional SDS-PAGE gel followed by Sypro Ruby staining. A representative gel is shown and full-length Highwire is marked with an arrowhead. Mass spectrometry identified one of the Highwire-interacting proteins as DFsn (approximately 27 kDa, indicated by arrow). (b) Schematic of DFsn protein (CG4643 in Drosophila) and its homologs in C. elegans, mouse and human. Green boxes denote the F-Box and blue boxes denote the SPRY domain. The overall amino acid identity to the DFsn protein is reported in the parenthesis on the right. (c) Larval brain lysate of either wild type (WT) flies or flies neuronally expressing His-Myc-tagged DFsn (HM-DFsn) was subject to co-immunoprecipitation (IP) with either anti-Highwire antibody (Hiw2b) or anti-Myc antibody (9E10). Both the input and the IP complexes were analyzed by western blots (WB) with anti-Myc or anti-Highwire antibody. (d-j) When expressed in the central nervous system, GFP-DFsn is localized to NMJs. The localization of GFP-DFsn at the synapses is analyzed by double staining with anti-GFP and synaptic vesicle marker DVGLUT (e-g), or periactive zone marker FasII (h-j). In (d), the presynaptic terminal of (e-g) was visualized by HRP (horseradish peroxidase) staining; the arrow indicates a preterminal axon.