Fig. 1: Ush2a^delG/delG protein is stable, developmentally regulated, and mislocalized.

A Developmental steady-state levels of total usherin transcripts are comparable in WT and Ush2a^delG/delG retinas. Shown are mean ± SEM from three independent samples. Statistical significance was determined by one-way ANOVA. P values: P15: p = 0.0043 (**). N value: 3 mice per time point and genotype and measurements were made in triplicates. B Immunoblotting shows that the mutant protein and GFP are expressed only in the KI retinas. C Immunoblot shows that the mutant protein is unable to form complexes through electrostatic interactions. D Immunoblot under reducing and non-reducing conditions shows that truncated usherin is unable to form a covalent complex (upper panel). PRPH2 was used as a positive control (lower panel). E Full-length usherin is located at the ciliary base (upper left and middle images) and OPL (lower left image). The truncated protein is absent from the P30 WT retina (right images). F Co-staining of retinal sections from P30 Ush2a^delG/delG for usherin and acTub shows the absence of full-length usherin (left and middle images). Labeling for FLAG reveals the truncated protein to be localized in IS (right images). G Co-labeling for FLAG and acTub at P30 shows a dispersed localization of the mutant protein in the IS, without distinct localization in the periciliary region. H Co-labeling for FLAG and STX3 demonstrates the accumulation of the truncated protein in the IS (left images). 3D-remodeling showing truncated usherin organized in compartments (right images). I–K Immunoblots of fractionated WT and Ush2a^delG/delG retinal extracts showing full-length usherin localized in the membrane fractions in the WT and absence in Ush2a^delG/delG retina (I). Immunoblot probed with anti-FLAG antibody revealed the major portion of the mutated protein is in the cytosolic fraction while a minor portion is present in the membrane fraction (K). GAPDH was used as a cytosolic marker, IRBP as a S-IPM marker, while PRPH2 was used as a membrane marker. L Immunoblot probed with anti-FLAG antibody showing the mutant protein is glycosylated like PRPH2. OS outer segment, IS inner segment, ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, S-IPM soluble inter photoreceptor matrix, Cyt. cytoplasm, Memb. membrane, Glyco. glycosylated, Deglyco. deglycosylated, Red. reducing, Non-Red. non-reducing.