Western analysis of sulfated proteins in retinal extracts from different species using the PSG2 antibody. A&C, Western blots using PSG2 antibody; B&D, sister gels stained with Coomassie Blue. Thirty micrograms of protein extracts were loaded in each lane. After reducing SDS-PAGE and transfer, the blot was probed with PSG2.

Sulfated proteins in the inter-photoreceptor matrix. A. Western blot of sulfated proteins in 661W conditioned medium. Thirty micrograms of mouse retinal protein extracts were loaded in the first lane. The other two lanes each contained concentrated proteins from 50 mL of media. 661W CM, 661W conditioned medium; un-CM, unconditioned regular medium. Gel was run under reducing conditions. The black asterisk denotes a protein band that is not observed in retinal extracts. The gray arrowhead points to the albumin present in the media. The black arrow points to a band that is a component of the fetal bovine serum medium. B. & C. Western blots to identify sulfated proteins in the inter-photoreceptor matrix (IPM) of the mouse retina. B. shows a Coomassie Blue stained gel and C shows a sister gel that was transferred to a membrane and probed with PSG2 antibody. S, soluble fraction; IS, insoluble fraction. IS-IPM⁺, contained the insoluble part of the IPM as well as the rest of the retina.

Western blot analysis for sulfated proteins in extracts from human retina, human RPE and the ARP-19 cell line. Thirty micrograms of extracts were loaded in each lane, the gel was run under reducing conditions and then transferred to a PVDF membrane. The blot was probed with PSG2. The arrow points to a protein species that may be present in all samples, albeit at much reduced levels in the human retina.

Detection of TPST-1 and TPST-2 transcripts in mouse and human retinas. A representative gel showing the DNA bands amplified in a RT-PCR reaction using cDNAs prepared against total RNAs from human and mouse retinas. Reactions were performed in triplicates on samples before (−RT) or after (+RT) reverse transcription. HPRT was used as a control, while PCR reactions without RT served as negative controls. Product sizes (bp), human TPST-1, 483; human TPST-2, 465; mouse TPST-1, 402; mouse TPST-2, 424. In the absence of reverse transcription (−RT). Primer dimers were observed in the Tpst2 lanes in the absence of reverse transcription.

Characterization of sulfated retinal proteins. A. Western blot (with PSG2) of sulfated proteins in wt mouse retina after treatment with PNGaseF. Thirty micrograms of proteins were loaded in each lane and gel was run under reducing conditions. B. Western blot of sulfated proteins in permanent cell lines representing different retinal cell types. Thirty micrograms of protein extracts were loaded in each lane. The asterisks denote proteins that are unique to the cell type and the arrow points to a sulfated protein that seems to be shared by all cell lines. 661W, a cone photoreceptor cell line; MC-1, a rat Müller cell line; RGC-5, a rat ganglion cell line. Mouse retina, first lane, was used as a reference. C. Western blot of sulfated proteins in wt and rd/rd mouse retinas. Thirty micrograms of proteins were loaded in each lane. The black arrowhead points to the common band; the black arrow points to a band that is only present in the rd/rd retina; and the asterisk points to the lower band that is only present in wt retinas. All blots were probed with PSG2.

Western blot analysis of sulfated proteins in mouse RPE and S-IPM. Fifty micrograms of protein extracts were loaded in each lane, the gel ran under reducing conditions and transferred to a PVDF membrane. The blot was probed with PSG2. Black asterisks denote bands unique to RPE; the white asterisk denotes a band that may be present in S-IPM but originated in the RPE; and the white arrowhead points to a band that is shared between RPE and S-IPM while little black arrows mark protein bands similar in retina and S-IPM.

Cellular localization of sulfated proteins in the human and mouse retinas. Sections of peripheral human retina were incubated in the absence (A) or presence (B&C) of PSG2 antibody. C. is the same image in B. without the DAPI staining of nuclei to better visualize the staining of the different layers of the retina with PSG2. The small white arrow in A is pointing to a blood vessel; the white arrowheads in B&C are pointing to either ganglion cell processes or inner limiting membrane and the green arrow in C is pointing to a junctional layer between OS and RPE that is heavily labeled. Inset in C is an enlargement of the area marked by the green arrow. All images were photographed at the same magnification and exposure time. Although the RPE seems to autofluoresce (A), there is more intense signal observed in the presence of PSG2 antibody (B&C). The asterisks are marking choroidal blood vessels. D&F, Nomarski images of wt mouse retinal sections showing the different layers. E. Fluorescent staining of a normal mouse retinal section with PSG2 antibody that recognizes sulfotyrosines on proteins. G. A section similar to that shown in E was incubated with PSG2 and 4 mM sulfotyrosine and imaged at the same settings as E. The specificity of the immunohistochemistry is confirmed by the lack of staining resulting from competition by sulfotyrosine. Similar result (as in G) is obtained when the primary antibody is eliminated. Sc, sclera; RPE, retinal pigment epithelium; OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.