Fig. 7. βV spectrin indirectly interacts with prestin. (A,B,D) In vitro binding assays. (A) Neither GST-tagged βVCH nor GST-tagged βVR29 directly interact with full-length prestin. (B) By contrast, the interaction between GST-tagged βVR29 and prestin can be detected when protein extracts derived from P90 organs of Corti (OC) are added to the binding solution. (C-E) Mapping of the βV spectrin-binding region on prestin and of the prestin-binding region on βV spectrin. (C) Schematic diagram of the transmembrane topology of prestin and its deletion constructs used in the pull-down assays. Prestin is a 10- to 12-transmembrane-domain protein, with relatively large cytoplasmic N and C termini. The numbers refer to the corresponding positions of amino acid residues in the rat prestin. (D) In the presence of P35 OC lysates, both the full-length prestin (prestin-FL) and the prestin C-terminal cytoplasmic domain (prestin-C) bind to either GST-tagged βVR29 or GST-tagged βVPH. (E) Western blot. In the reciprocal experiment, GST-tagged prestin-C529 (aa 529-744) binds to the myc-tagged βVR26 (aa 3012-3674) only in the presence of OC lysates.