For preparation of crude membranes, homogenates were spun at 700 for 10 min at 4C, pellets washed with 1 volume of homogenization buffer, spun again as above, supernatants were pooled then spun at 100 000 for 1 h at 4C and pellets resuspended in cold 50 mM TrisCHCl, pH 7.4, 150 mM NaCl, 1 mM XL-228 EDTA, 1% Triton X-100. expressing internally deleted forms of PrP or wild-type (wt)Dpl within the CNS. The presence of Dpl in the brain of Rabbit Polyclonal to PKA-R2beta (shadow of the prion protein’), is present from zebrafish to humans and is predicted to encode a short protein, Shadoo (Sho) (Premzl is located on chromosome 7 in mice, away from the gene complex on chromosome 2. Building on the genetic interaction between PrPC and Dpl or PrP, we have established an assay for PrPC activity in primary cultures of cerebellar granule cells (Drisaldi open reading frame present in genomic DNA of species from mammals to fish (Premzl gene (Makrinou expression by RTCPCR (Premzl (Figure 2A) and recognizes an N-terminal epitope contained within residues 30C61 (Supplementary Figure S1). Assessed by Western blot of tissue lysates, 06rSH-1 was virtually devoid of cross-reactive species (Supplementary Figure S1). Cross-reactive species of molecular weights incompatible with authentic Sho were present in analyses with antisera 04SH-1 and with 06SH-3, but these had varying intensities and/or different molecular weights for the two antisera. Consequently, the following comments are restricted to signals detected by two or more varieties of -Sho antibodies. Open in a separate window Figure 2 Analysis of recombinant Sho in and expression of murine Sho in cultured cells. (A) Schematic representation of the Sho protein. The location of the mapped epitopes for -Sho peptide antisera (04SH-1 and 06SH-3) and -recombinant Sho (06rSH-1) are shown. (B) Circular dichroism spectrum of recombinant mouse Sho, rSho(25C122). The spectral trace is consistent with a random coil configuration. (C) Cell surface expression of wt Sho and a mutant Sho allele lacking the hydrophobic tract in non-permeabilized transfected N2a cells as demonstrated by immunocytochemistry. Scale bar, 50 microns. (D) Diminution of Sho signal in the cell lysates of Sho-transfected N2a cells following pretreatment with increasing concentrations of PI-PLC. (E) Western blot showing expression of a wt Sho transgene in N2a cells with or without PNGaseF treatment. A lysate from cells transfected with empty vector is included to show antibody specificity. Similar to PrPC, murine Sho is revealed as being expressed at the cell surface, XL-228 hybridization using antisense-strand riboprobes prepared against the mouse open reading frame (but not sense-strand controls) yielded signals in the adult mouse CNS. Analyses of the hippocampus and cerebellum revealed prominent signals in the cell bodies of pyramidal cells and Purkinje cells, respectively (Figure 4B and ?andJ).J). By way of comparison, has a broader pattern of neuronal expression (Kretzschmar hybridization (Figure 4D and ?andL),L), that is, hippocampal neurons and cerebellar Purkinje cells. In the case of antisera 04SH-1, these signals were absent when antibodies were preincubated in a solution containing the Sho86C100 peptide immunogen (Figure 4C and ?andK).K). Besides defining Sho as the second’ cellular prion protein present in neurons of the adult CNS, these data define intracellular transport phenomena, as immunohistochemical signals were present in cell processes in addition to the cell bodies detected by antisense riboprobes (i.e., predicted to contain Sho mRNA). In the case of Purkinje cells, immunostaining was present not only in cell bodies but also prominent in their processes, specifically in the dendritic arborizations present within the molecular layer of the cerebellum (Figures 4L and 5FCH, signals detected with all three antisera). A related phenomenon was observed in the hippocampus, notably in CA1 pyramidal neurons. Here, Sho immunoreactivity was absent from axonal projections (with all three -Sho antibodies), present in cell bodies (seen by all three -Sho antibodies), and notable in the apical dendritic processes located in the stratum radiatum of the hippocampus (strong signals with 04SH-1 and 06SH-3, and a less intense signal with 06rSH-1) (Figures 4D and 5ACC). Open in a separate window Figure 4ah Localization of mRNA and Sho protein in the adult mouse brain. (ACH) The hippocampus, and (ICP) the cerebellum. wt C57/B6 mice are presented in all sections, with the exception of B6 congenic hybridization: panels A, B, and I, J represent hybridizations with Sho sense-strand (A, I) or antisense (B, J) cRNA probe. Sections are not counterstained and blue staining from NBT/BCIP substrate represents hybridization to mRNA. Immunohistochemistry: all other panels of mouse brains with genotypes as noted above. Anti-Sho antibody 04SH-1 (-Sho) antibody was used with (C, K) or without (D, L) preincubation with Sho(86C100) peptide. Antibodies 7A12 and 3F4 were used for the detection XL-228 of mouse PrP (E, F, M, N) and hamster PrP (G, H, O, P), respectively. Note the Sho staining of CA1 apical dendritic processes (D, black bracket) and Purkinje cell layer (L, white arrow), and absence of Purkinje cell-body staining with -moPrP (N) and relative.
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