Supplementary MaterialsAdditional file 1 Single confocal z-sections of rods expressing Crb2a transgenes. rods. (C-C”‘) Crb2aExtra_TM transgenic rods. (D-D”‘) Crb2aExtra_Secr transgenic rods. (E-E”‘) Crb2aIntraWT transgenic rods. (F-F”‘) Crb2aIntraFBD transgenic rods. (G-G”‘) Crb2aIntraPBD transgenic rods. (H) Western blot of 5 d wild-type zebrafish labeled with anti-GOLGA5 antibodies reveals a single protein of the expected molecular weight. The western blot was performed as described in , anti-GOLGA5 (Sigma HPA000992) was used at 1:1000 and HRP-conjugated goat anti-rabbit was used at 1:30,000. Scale bar, 5 m. 1471-2121-11-60-S3.JPEG (782K) GUID:?62E9C8A1-90B3-4E5D-AD03-F0F7B0AFA6E3 Additional file 4 Effects of Crb2a construct expression on Moe localization. (A-H”) Confocal z-projection of 6 d photoreceptor layer labeled with anti-GFP (green), anti-HA antibodies (red) and anti-Moe antibodies (blue). (A-A’) Wild-type rods. (B-B”) Crb2aIntraWT transgenic rods. (C-C”) Crb2aIntraFBD transgenic rods. (D-D”) Crb2aIntraPBD transgenic rods. (E-E”) Rabbit Polyclonal to CAMK5 Crb2aIntraFBDPBD transgenic rods. (F-F”) Crb2aFL transgenic rods. (G-G”) Crb2aExtra_TM transgenic rods. (H-H”) Crb2aExtra_Secr transgenic rods. Scale bars, 5 m. 1471-2121-11-60-S4.JPEG (1019K) GUID:?95DB2BB5-CEFC-45C2-88B3-8632005A22E6 Additional file 5 Effects of Crb2a construct expression on Prkci localization. (A-H”‘) Confocal z-projection of 6 d photoreceptor layer labeled with anti-HA (red), anti-Prkci antibodies (blue) and anti-GFP labeling (green). (A-A”) Wild-type photoreceptor rods. (B-B”‘) Crb2aIntraWT transgenic rods. (C-C”‘) Crb2aIntraFBD transgenic rods. (D-D”‘) Crb2aIntraPBD transgenic rods. (E-E”‘) Crb2aIntraFBDPBD transgenic rods. (F-F”‘) Crb2aFL transgenic rods. (G-G”‘) Crb2aExtra_TM transgenic rods. Apremilast inhibition (H-H”‘) Crb2aExtra_Secr transgenic rods. Scale bars, 5 m. 1471-2121-11-60-S5.JPEG (1.2M) GUID:?87FFF5A1-1864-4AC4-89D5-267AC06C7D4A Additional file 6 Western blot of 6 d wild-type (nonTg WT), Crb2aIntraWT, Crb2aIntraFBD, Crb2aIntraPBD, Crb2aIntraFBDPBD, Crb2aFL, Crb2aExtra_TM, Crb2aExtra_Secr transgenics probed with anti-HA antibodies. Western blotting was performed as previously described  and probed with anti-HA (clone 16B12) and HRP-conjugated goat anti-mouse. The predicted molecular weight Apremilast inhibition of Crb2aIntraWT protein is 11 kD (retaining the signal peptide) but on western blot the major Crb2aIntraWT protein is ~30kD, suggesting that it may be post-translationally modified or forms homoligomeres. Despite trying multiple gel and transfer conditions we were unable to detect Crb2aIntraFBD, Crb2aIntraPBD, Crb2aIntraFBDPBD proteins, which by immunohistochemistry are expressed at similar levels as Crb2aIntraWT. It is possible that Crb2aIntraFBD, which is be predicted to be about the same Apremilast inhibition molecular weight as Crb2aIntraWT, is not post-translationally modified or does not dimerize and, thus, is too small, like Crb2aIntraPBD and Crb2aIntraFBDPBD with predicted molecular weights ~8.8kD (with signal peptide) to be captured by Western blot analysis. 1471-2121-11-60-S6.JPEG (236K) GUID:?B8B6F68A-59AC-4438-A849-92BD3285021B Abstract Background Vertebrate retinal photoreceptors are morphologically complex cells that have two apical regions, the inner segment and the outer segment. The outer segment is a modified cilium and is continuously regenerated throughout life. The molecular and cellular mechanisms that underlie vertebrate photoreceptor morphogenesis and the maintenance of the outer segment are largely unknown. The Crumbs (Crb) complex is a key regulator of apical membrane identity and size in epithelia and in Drosophila photoreceptors. Mutations in the human gene em CRUMBS HOMOLOG 1 /em ( em CRB1 /em ) are associated with early and severe vision loss. Drosophila Crumbs and vertebrate Crb1 and Crumbs homolog 2 (Crb2) proteins are structurally similar, all are single pass transmembrane proteins with a large extracellular domain containing multiple laminin- and EGF-like repeats and a small intracellular domain containing a FERM-binding domain and a PDZ-binding domain. In order to begin to understand the role of the Crb family of proteins in vertebrate photoreceptors we generated stable transgenic zebrafish in which rod photoreceptors overexpress full-length Crb2a protein Apremilast inhibition and several other Crb2a constructs engineered to lack specific domains. Results We examined the localization of Crb2a constructs and their effects on rod morphology. We found that only the full-length Crb2a protein approximated the normal localization of Crb2a protein apical to adherens junctions in the photoreceptor inner segment. Several Crb2a construct proteins localized abnormally to the outer segment and one construct localized abnormally to the cell body. Overexpression of.