(A) Two protein isoforms are generated from alternatively spliced transcripts of Myo15. Transcripts incorporating exon 2 encode isoform 1 (396 kDa; Genbank: NM_010862.2), while exclusion of this exon produces isoform 2 (262 kDa; Genbank: NM_182698.2). Both isoforms have identical motor and tail domains, including a PDZ ligand, SH3, MyTH4 and FERM moieties. The mutant Myo15 alleles used in this study are shown along with antibody epitopes. (B) Auditory brainstem response (ABR) thresholds at 4, 20 and 48 kHz for Myo15+/+, Myo15+/ΔN and Myo15ΔN/ΔN mice at 6 weeks of age. Data are mean ± SD (n = 3–6 animals per group). (C) ABR thresholds at 20 kHz, measured from 2, 4 and 6 weeks old Myo15+/+, Myo15+/ΔN and Myo15ΔN/ΔN mice. Data are mean ± SD (n = 3–6 animals per group). (D) Distortion product otoacoustic emission (DPOAE) levels (2F1-F2) at 12 kHz in Myo15+/+ and Myo15ΔN/ΔN mice at 6 weeks. Data are mean ± SD (n = 3–4 animals per group). (E–G) Relative expression of Myo15 isoforms in wild-type cochleae measured with RT-qPCR at ages indicated. Probes target exon junction 13–14, detecting the motor domain common to both isoforms 1 and 2 (E); exon junction 2–3, detecting isoform 1 (F); exon junction 1–3, detecting isoform 2 (G). Relative expression 2^(-∆∆CT) for each Myo15 transcript was normalized first to the housekeeping gene (Tbp) and then to the respective isoform expression at P0. The total expression of both isoforms remains stable (E), however there is a transition from isoform 2 to isoform 1, which becomes the dominant mRNA species by P21 (see Figure 1—figure supplement 1D). Data are mean ± SD (n = 3–5 biological replicates per condition). Asterisks indicate significance: n.s., p > 0.05; **, p < 0.01; ****, p < 0.0001 (ANOVA with Tukey's multiple comparison test). (H) Identical qPCR probes were used to assay Myo15 expression in Myo15+/ΔN and Myo15ΔN/ΔN cochleae at P0. Relative expression 2^(-∆∆CT) values were normalized to Tbp and then to expression in heterozygous Myo15+/∆N samples. Data are mean ± SD (n = 3–4 biological replicates per condition). n.s., p > 0.05 (t-test of independent variables).