Fig. 2.

Ablation of the Ush2a gene expression in mice. For details of targeting strategy, see supporting information (SI) Methods. (a) Immunoblotting run on agarose gels and probed by either α-Usherin-N and α-Usherin-C antibodies showed complete ablation of the long usherin variant (estimated at 600 kDa) in the homozygous mutant. Dystrophin from skeletal muscles was shown as a reference for molecular mass. The same samples run on a polyacrylamide gel and probed γ-tubulin served as a loading control. (b) RT-PCR analysis of WT tissues showed that usherin transcripts originated from the neural retina, not RPE cells. The expected 1.2-kb PCR product was absent from homozygous mutant tissues. (c) Immunofluorescence for usherin in the retina. In WT retinas, both α-Usherin-N and α-Usherin-C recognized a dotted staining (green) pattern between the inner and outer segment, indicative of the connecting cilia (arrowhead). In the Ush2a^−/− retina, this staining pattern was missing. Nuclei were counterstained blue with Hoechst dye 33342. IS, inner segment; OS, outer segment; ONL, outer nuclear layer.