To determine whether the absence of CaBP4 can affect the level of Unc119 protein, Unc119 was analyzed in the retinas of CaBP4-knockout mice (
Cabp4 −/−). To isolate and enrich in proteins localized to the outer plexiform layer, the method described by the Arshavsky group,
25 which combines serial tangential sectioning of flatmounted frozen retinas with Western blot analysis, was used. Serial sectioning of retinas was performed on frozen retinas with the photoreceptor side up. The collected fractions included approximately two thirds of the retina from the photoreceptor side. Serial sections were obtained from either 3
Cabp4 +/+ or 3
Cabp4 −/− mouse retinas to compare the protein content of multiple synaptic markers. The distribution of Unc119 in all retinas was analyzed using PKCα as a marker protein. PKCα is expressed throughout the rod bipolar cells, from the axon terminals in the inner nuclear layer to their dendrites that form a dense network in the OPL, where they contact photoreceptor pedicles
26 (Fig. 6A) . The distribution of PKCα was analyzed, together with the distribution of Unc119 or other synaptic proteins, to monitor the reliability of the section series and the relative amounts of the studied synaptic proteins to PKCα in the OPL. The distribution of CaBP4 was found to overlap the distribution of Unc119
(Fig. 6A) . The distributions of rhodopsin, syntaxin 3, and PSD95 were also analyzed in all fractions. Because there is always some variation inherent to the Western blot analysis, samples can only be compared if loaded on the same Western blot. Therefore, and because of the limited amount of material obtained using this method, fractions 3 to 5, which contained the higher amounts of Unc119 in all retinas
(Fig. 6A) , were combined and used to compare the level of Unc119 with other synaptic proteins.
Figure 6Bshows that the amount of Unc119 protein in CaBP4-knockout mice was lower than that in wild-type mice, though no differences were observed for PKCα marker proteins in the same fraction. As expected, CaBP4 was not detected in fractions from
Cabp4 −/− mice. The amount of Unc119 protein was also compared with the amount of other photoreceptor synaptic proteins (i.e., PSD-95 and syntaxin 3).
27 Similar amounts of syntaxin 3 were detected in synaptic fractions of
Cabp4 +/+ and
Cabp4 −/− mice, in contrast to Unc119
(Fig. 6B) . PSD-95 was also reduced in
Cabp4 −/− mice, but Unc119 was still more severely decreased
(Fig. 6B) .