Danio rerio prion protein 2: AAS00160 Sparus aurata prion protein 1 coding sequence: ABB90540 Supporting Information Table S1 Percentage amino acid sequence homology between prion proteins of different species. Full sequences were aligned. The NCBI accession numbers of sequence data are: Homo sapien (human), AAC78725; Bos Taurus (cow), AAD19998; Ovis aries (sheep), CAE00188; Mus musculus (mouse), AAH06703; Meso- cricetus auratus (hamster), AAA37092; Takifugu rubripes (fugu), AAN38988; Danio rerio (zebrafish) prion protein 1, AAS00159; Danio rerio prion protein 2, AAS00160; Sparus aurata (gilthead sea bream) prion protein 1, ABB90540. Sequence alignments were performed by ALIGN (version 2, Myers and Miller, CABIOS (1989) 4:11–17). Found at: doi:10.1371/journal.pone.0006175.s001 (0.09 MB TIF) Table S2 Cumulative record of the number of fish maintained post challenge. A, Fish inoculated with either scrapie- or normal ovine brain homogenate. B, Fish inoculated with either BSE- or normal bovine brain homogenate. Found at: doi:10.1371/journal.pone.0006175.s002 (0.04 MB DOC) Table S3 Cumulative record of brain tissue samples examined. A, BSE-challenged and bovine control fish samples. B, Srapie- challenged and ovine control fish samples. Found at: doi:10.1371/journal.pone.0006175.s003 (0.14 MB DOC) Table S4 Cytoanatomical analysis of brains from BSE-chal- lenged fish sacrificed 24 months post challenge. The deposits have been classified into 2 morphological categories. F, fibrillary, .10 mm in diameter; NF, non fibrillary, circular,10 mm in diameter. Plus and minus symbols indicate the abundance of deposits: 2, 0; +, 1–5; ++, 6–15; +++, 16–50, ++++, .50. NP, anatomical region not present in section; Mol L, molecular layer; Gran L, granular layer; WM, white matter; Cx, cortex; Ce, cerebellum; Vc, valvula cerebelli; Tel, telencephalon; Di, dien- cephalon; OT, optic tectum; Br. st., brain stem. Found at: doi:10.1371/journal.pone.0006175.s004 (0.13 MB TIF) Figure S1 Comparison of antibody specificities for the PrPs of gilthead sea bream, cow and sheep by western blot analysis. A, Five 0.4 mg brain equivalent-aliquots of gilthead sea bream brain homogenate were loaded onto a 12% SDS-PAGE gel. B, Alternating lanes of a 12% SDS-PAGE gel were loaded with 3 mg tissue equivalents of PrPSc-enriched (see Materials and Methods) bovine BSE brain homogenate (lanes 1, 3, 5, 7 & 9) and ovine scrapie brain homogenate (lanes 2, 4, 6, 8 & 10). The electrophoretically separated proteins were transferred to PVDF membranes that were cut into four sections (in B, each section included two adjacent lanes). Each section was stained with one of five primary antibodies: 6H4 (1:5000; lanes A1, B1, B2); FuguPrP1 (1:20000; lanes A2, B3, B4); ZebPrP1 (1:10000; lanes A3, B5, B6); ZebPrP2 (1:35000; lanes A4, B7, B8); SaurPrP1 (1:20000; lanes A5, B9, B10). After incubation with the appropriate alkaline- phosphatase-conjugated secondary antibody, the blots were developed with the CDP-Star reagent. The arrow heads indicate the positions of the molecular mass markers: A, 62 kDa and 47.5 kDa; B, 32.5 kDa. Found at: doi:10.1371/journal.pone.0006175.s005 (0.28 MB TIF) Figure S2 Antibody specificity in IHC. Sagittal, 4 mm-thick serial brain sections from control gilthead sea bream were treated immunohistochemically with four different primary antibodies, TSE Transmission to Sea Bream without proteinase K digestion. A, SaurPrP1 (1:2000); B, ZebPrP2 (1:2000); C, Pre-immune serum from the rabbit in which SaurPrP1 was raised (1:2000); D, PrP-specific immunoglobulin- depleted SaurPrP1 (1:2000). Arrowheads indicate the existence (A, B) or absence (C, D) of PrP-immunopositivity. SGP, striatum griseum periventriculare; SFP, striatum fibrosum profundum; SGC, striatum griseum centrale; SPFE, striatum plexiforme et fibrosum externum; SFM, striatum fibrosum marginale; ML, molecular layer of the valvula cerebelli; GL, granular layer of the valvula cerebelli. Scale bars, 100 mm. Found at: doi:10.1371/journal.pone.0006175.s006 (8.23 MB TIF) Figure S3 Temporal observation of the brains from control fish challenged with normal ovine brain homogenate. Sagittal brain sections taken at 12 and 24 months p.i. from fish challenged with normal ovine brain homogenate were stained with H&E (A, D), or immunolabeled with SaurPrP1 (1:2000) without PK-digestion (B, E) and with PK-digestion (C, F). Images show diencephalon. The mean number of deposits (per section of fish containing deposits) observed in different brain regions without PK-treatment is indicated by the fill-type in the schematic drawings at the far left. Abbreviations as in Figure 2 of the main manuscript. The following areas were not examined: OB; OIN; OC; P. Rectangles indicate areas of magnification shown in the panels directly below. Scale bars, 100 mm. Found at: doi:10.1371/journal.pone.0006175.s007 (3.00 MB TIF) Figure S4 Temporal observation of the brains from control fish challenged with normal bovine brain homogenate. Sagittal brain sections taken at the indicated timepoints p.i. from fish challenged with normal bovine brain homogenate were stained with H&E (A, D, G), or immunolabeled with SaurPrP1 (1:2000) without PK- digestion (B, E, H) and with PK-digestion (C, F, I). Images show diencephalon. The mean number of deposits (per section of fish containing deposits) observed in different brain regions without PK-treatment is indicated by the fill-type in the schematic drawings at the far left. Abbreviations as in Figure 2 of the main manuscript. The following areas were not examined: OB; OIN; OC; P. Rectangles indicate areas of magnification shown in the panels directly below. Scale bars, 100 mm. Found at: doi:10.1371/journal.pone.0006175.s008 (4.48 MB TIF) Figure S5 Sensitivity to proteinase K treatment of TSE- challenged sea bream brain tissues 24 months p.i.. After a short purification treatment, 0.4 mg brain equivalents from brain homogenates of either scrapie- (lanes 1, 3, 5 & 7) or BSE- challenged (lanes 2, 4, 6 & 8) fish, were digested with increasing proteinase K concentrations (0 mg/ml, lanes 1 & 2; 0.1 mg/ml, lanes 3 & 4; 1 mg/ml, lanes 5 & 6; 10 mg/ml, lanes 7 & 8) for 1 hr at 37uC. The samples were analyzed on a 12% SDS-PAGE gel, then electrotransferred onto a PVDF membrane and probed with SaurPrP1 polyclonal antibody (1:20000). After incubation with the appropriate secondary antibody, the immunoblots were finally developed with the ECL western blotting substrate. Arrowhead, 47.5 kDa. Found at: doi:10.1371/journal.pone.0006175.s009 (0.27 MB TIF) Text S1 Statistical analysis of data derived from the scrapie - challenged group. Found at: doi:10.1371/journal.pone.0006175.s010 (0.02 MB DOC) Acknowledgments The authors are thankful to Dr. N. Grigoriadis’ group at AHEPA Hospital of Thessaloniki for their support with this microscopy project; Drs. H. Budka and G. Kovacs at the General Hospital of Vienna for their expert PLoS ONE | www.plosone.org 12 July 2009 | Volume 4 | Issue 7 | e6175