Analysis of the Nedd4-binding protein 3 domain structure and characterization of an affinity-purified polyclonal Nedd4-binding protein 3 antibody. (A) Schematic overview of the Nedd4-binding protein 3 (N4BP3) domain structure in Rattus norvegicus. The Nedd4 PY-binding domain (aa 239 to 242, PPPY), coiled-coil (CC; aa 295 to 483) and Fez1 (aa 359 to 520) domains are highlighted. Sequence information of the CC and Fez1 domains derives from the Pfam 26.0 database (http://pfam.sanger.ac.uk/). (B) Homology analysis of N4BP3 primary structure (amino acids, full-length protein (overall), PY (PPPY), CC and Fez1 domains). Note the 100% homology within the Nedd4-interacting PY domain between species. (C) To characterize the affinity-purified polyclonal N4BP3 antibody, green fluorescent protein (GFP)-N4BP3 and Myc-N4BP3 were expressed in HEK-293T cells. Both fusion proteins were biochemically detected at their predicted molecular weights (70 vs. 100 kDa) in Western blot analysis by either the N4BP3 antibody or GFP and Myc antibodies, respectively. Furthermore, the affinity-purified antibody detects both in vitro translated Myc-N4BP3 (transcription/translation (TNT) assay) and endogenous N4BP3 in lysate from DIV7 primary hippocampal cultures at approximately 70 kDa. (D) GFP-N4BP3 (Alexa Fluor 488 dye (Molecular Probes/Invitrogen, Eugene, OR, USA), green) is expressed in dotlike structures localized all throughout the cytosol, excluding the nucleus. The same subcellular structures are immunodetected by the N4BP3 antibody (Alexa Fluor 568 dye (Molecular Probes/Invitrogen), red). See insets for clear overlay of green and red channels.