The fact that vision is also affected and that the retina is devoid of stereocilia-like structures led us to examine Usher protein function at the ribbon synapses, highly Dasatinib specialized structures present in both photoreceptors and hair cells, where many of the Usher proteins have been localized. A developmental survey for Usher protein expression focused our initial GDC-0941 studies on CDH23, PCDH15 and VLGR1. We developed and characterized antibodies for these three Usher proteins. Due to the presence of multiple isoforms generated by alternative splicing and/or by the use of alternative promoters and splice sites, most of the Usher mouse models available are not true knockouts but spontaneous or induced mutants that either lack some of these isoforms or express dysfunctional proteins. Just recently, the expression of several PCDH15 isoforms in the stereocilia were described using the Ames waltzer mouse model av6J, that carries a presumptive inframe deletion in exon 22. The use of antibodies directed against the three different cytoplasmic domains of PCDH15 demonstrated normal immunostaining at the stereocilia. However the use of an extracellular domain-directed antibody showed a marked decreased in PCDH15 expression, demonstrating that the selection of the domain used to raise antibodies is a critical step for isoform detection. This concept is further validated by our demonstration of apical stereociliary immunostaining for VLGR1 hair cells isolated from P30 mice, which suggests that a previously undescribed isoform of VLGR1 containing the EAR domain, but not the carboxylterminal domain is present in mature hair cell stereocilia. Therefore, to establish the isoform specificity of our antibody preparations, we used two different approaches: 1) western blot analysis of P3 inner ear and neuroretina with the affinity purified primary antibody or the flow through following preadsorbtion of antibodies with immobilized peptide immunogen and 2) transient knock down studies of the Usher transcripts in differentiated UB/OC-1 cells using siRNAs derived from the DNA sequences comprising the peptide immunogens. Bands observed on western blots using affinity purified antibodies were markedly reduced or absent when duplicate blots were probed with the flow through following affinity purification on immobilized peptide immunogens. UB/ OC-1 cells showed differences in the efficiency of knockdown for all the Usher transcript isoforms within a given Usher protein with a reduction in expression between 20% to 100%, likely due to differences in the efficacy of the siRNAs employed. We were able to establish a spatiotemporal pattern of expression for the Usher proteins at the apical and basal aspects of the hair cells and in the neuronal terminals, between P1 and P14.