Figure 1.

Generation of α₂δ-4 KO mice. A, Schematic illustrating domain structure of α₂δ-4 and strategy for interrupting exon 2 using the indicated guide RNA sequences (sgRNA 1f and 1r). The four chemosensory domains (1–4) are indicated in colored boxes. VWA, von Willebrand A domain. Numbers beneath diagram indicate amino acids. Putative disulfide bonds (S-S) between α₂ and δ are shown above the diagram. The resulting amino sequence change (underlined) and premature truncation are shown below. B, PCR with primers flanking the deleted region in exon 2 using retinal genomic DNA from WT, KO, and HET mice. C, D, Western blots probed with α₂δ-4 or HA antibodies of lysates from HEK293T cells untransfected (control) or transfected with HA-tagged α₂δ variants (C) or retina from WT, HET, or KO mice (D). Arrow and asterisks indicate specific and nonspecific protein detection, respectively. E, F, Confocal micrographs showing labeling with α₂δ-4 antibodies in WT and α₂δ-4 KO mouse retina at P21 (E) and macaque and human retina (F). E, Mouse retina was double-labeled with fluorescent PNA to mark cone terminals. α₂δ-4 labeling is present at ribbons of rods (arrows) and cones (arrowheads). Scale bars, 2 μm.