Fig 2: Hearing impairment is inversely correlated with Nptn-dependent localisation of PMCA in outer hair cell stereocilia.(A) Immunolabelling of cochlear cryosections using anti-PMCA1 and anti-PMCA2 antibodies. In wild type tissue, PMCA1 is predominantly localised to the membrane of IHCs and OHCs, whereas PMCA2 is localised to OHC stereocilia and lateral supporting cells. Labelling of the PMCAs at these locations was substantially reduced in Nptntm1b/tm1b cochleae. Scale in (A) = 20 µm. (B) The anti-pan-PMCA antibody detects PMCA1-4. Maximum intensity projections of whole-mount cochlear immunolabelling using the anti-pan-PMCA antibody showed labelling of OHC stereocilia and membrane of IHCs in Nptn+/+ mice, which were substantially reduced, but not absent, in Nptntm1b/tm1b cochleae. (C) Using the anti-pan-PMCA antibody, no labelling differences were seen in Nptn?exon2/?exon2 cochleae compared to wild-type. Images in (A), (B), and (C) taken from the mid-apical region of the cochlea. Scale in (B) and (C) = 10 µm. (D) ABR threshold and DPOAE response measures from two Nptnfl/fl;Prestin-CreERT2+ mice following tamoxifen-induced cre-mediated recombination, with corresponding anti-pan-Np and anti-pan-PMCA immunolabelled mid-apical cochlear whole mounts (maximum intensity projections). Data from the individual mice are shown (red triangles) compared against wild type controls (black circles, mean ± SD). Due to varying levels of induction, the auditory phenotype exhibited was variable with some mice showing no significant phenotype (e.g. Mouse 1), while others showed increased ABR thresholds and a decreased DPOAE response (e.g. Mouse 2). The auditory phenotype was directly correlated to the level of Nptn recombination as demonstrated by immunolabelling using the anti-pan-Np antibody. Bundles that were strongly stained for Neuroplastin also had strong labelling for anti-pan-PMCA antibody (arrowhead), whereas absent Neuroplastin was correlated with a weak PMCA signal (*).