Allelic derivation and semiquantitative analysis of protease-resistant and protease-sensitive pathological prion protein species in P102L GSS cases.

<p>(*)in the upper part of the gel a smear is observed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032382#pone-0032382-g005" target="_blank">figure 5D</a>). Scores: − (negative); +/− (faint); + (weak); ++ (moderate); +++ (intense).</p

Immunohistochemical analysis of allelic prion protein species in P102L GSS.

<p>N.A. Not available. Scores: − (negative); + (light); ++ (mild); +++ (moderate); ++++ (intense).</p

Fractionation of mutant and wild-type PrP^Sc aggregates in GSS P102L.

<p>Brain homogenates from frontal cortexes of case # 5 (A, B) and case # 8 (C, D) were sedimented in a 10 to 60% sucrose gradient. After sedimentation, half samples were digested with PK (E, F, G, H). (A) PrP from case # 5 is distributed across all fractions albeit concentrated at the top (1–4) and at the bottom fractions (8–11); two bands of 11 and 8 kDa co-localize with PrP^Sc in fraction 11. (B) Immunoblot with ICSM-35 of case # 5 shows lower amounts of PrP aggregates, with a distribution similar to that observed with 3F4, in the absence of lower molecular species. (C) In case # 8, 3F4-reactive PrP was mainly distributed at the top and at bottom fractions, whereas ICSM 35 showed very low amount of PrP in the bottom fraction (D). (E) Mutant PrP aggregates in case # 5 were resistant to PK digestion; PK-resistant PrP^Sc species were more abundant in bottom fractions, containing also high amounts of the a smear spanning 8–12 kDa. (F) ICSM 35 stained PK-resistant PrP^Sc from case # 5 were detected only in the bottom fractions. (G) In case # 8, PrP27-30 is absent, while an 8 kDa fragment is detected at the fractions 9–11; on the contrary, no wild-type PrP species are seen (H).</p

Diagram of PK-resistant and PK-sensitive PrP^Sc molecules detected in GSS with the P102L mutation.

<p>PrP^Sc species originating from the mutant allele are depicted in orange, whereas wild-type PrP^Sc molecules are depicted in green. PrP8 is an internal PK-resistant fragment spanning residues 74/90 to 146/153. PrP27-30 is the main C-terminal PK-resistant PrP fragment generated by PK cleavage between PrP^Sc residues 74 and 103. PrP-CTF 16/17 and PrP-CTF 12/13 are smaller PK-resistant fragments whose allelic origin remains undetermined (depicted in blue).</p

Two-dimensional analysis of mutant and wild-type PrP27-30, C-terminal truncated fragments, and insoluble PrP^Sc species in GSS P102L.

<p>(A) Immunoblot with 3F4 of PrP27-30 in case # 4 shows three sets of differently glycosylated charge isomers migrating between 30 and 20 kDa, in addition to three spots with a mass of 8 kDa and pIs from 8.4 to 9.6 (arrow). (B) After deglycosylation, the 3F4-reactive core fragment resolves as five spots with net charges between 6.1 and 8.1. (C) ICSM 35 antibody decorates three major spots at 8.1, 7.7, 7.3, and a minor spot at 6.8. (D) Proteinase K-resistant PrP^Sc probed with 3E2 shows the three sets of PrP27-30, in addition to acidic isoforms migrating at 20 and 14 kDa, which after deglycosylation (E), resolved as a core fragment of six spots at 20 kDa, and C-terminal truncated species of 16–17- and 12–14-kDa. (F) In contrast, the PrP27-30 core fragment associated to E200K mutation resolves as a major spot at pI of 6.8. Immunoblots of the detergent-insoluble fraction, show the presence of naturally occurring PrP^Sc species with migration overlapping PrP27-30, the 8 kDa fragment (G), and C-terminal truncated fragments (H). (I) Schematic diagram of PK-resistant mutant and wild-type PrP^Sc forms.</p

Protease-sensitive PrP^Sc conformers in GSS P102L brain tissues.

<p>(A) Immunoblot with 3F4 of brain homogenate from case # 2 digested with PK before (lane 1) and after PNGase treatment (lane 2); lanes 3 to 6 show products of ‘cold PK’ (cPK)-treated homogenates, before (lanes 3 and 5) and after (lanes 4 and 6) PNGase treatment; in lanes 5 and 6 samples were NaPTA-precipitated; (B) cPK-treated (lanes 7–10) and NaPTA precipitated products (lanes 9 and 10) were revealed with ICSM 35, prior (lanes 7 and 9) or after deglycosylation. (C) Immunoblot with 3F4 of brain extracts from case # 8, after conventional PK treatment, shows a barely detectable 8 kDa band (lanes 1,2, arrow); in contrast, after cPK digestion (lanes 3 to 6) and NaPTA precipitation (lanes 5 and 6) consistent amounts of PrP27-30 and of the 8 kDa fragment are seen (lane 3–6) in PNGase untreated (lanes 3 and 5) or treated (lanes 4 and 6) preparations. (D) In case # 8, immunoblot with ICSM 35 did not reveal PrP^Sc after cPK treatment (lanes 7 and 8), wheras NaPTA precipitation showed a smear migrating in the upper part of the gel in a 50–70 kDa zone (lane 9, arrow), partially decreased by PNFase treatment (lane 10).</p

MOESM5 of Remodeling of nuclear landscapes during human myelopoietic cell differentiation maintains co-aligned active and inactive nuclear compartments

Additional file 5. Comparative topology of SC35 and RNA Pol II Ser5P, markers for transcriptional activity in relation to chromatin density maps. (A) 3D-SIM light optical mid-sections from whole 3D acquisitions of nuclei and representative inset magnifications delineating DAPI stained DNA (gray), immuno-stained SC35 (green) and RNA Pol II Ser5P (red). SC35, an integral part of splicing speckles is seen almost exclusively in the IC compartment while RNA Pol II Ser5P shows a preferential localization at decondensed chromatin sites or at the surface of compacted chromatin domain clusters (compare additional file 4). Scale bars: 2 µm; insets 0.5 µm (B) graphs highlighted with yellow background: relative signal distribution of SC35 (green) and RNA Pol II Ser5P (red) within respective DAPI defined DNA intensity classes. p < 0.001 for DAPI vs. SC35 and RNA Pol II Ser5P, and for SC35 vs. RNA Pol II Ser 5P, except for SC35 vs. RNA Pol II Ser5P in granulocytes (p = 0.004). Graphs highlighted with light-blue background: quantified levels of relative enrichment (positive values) or depletion (negative values) of SC35 (green) and RNA Pol II Ser5P (red) signals relative to the DAPI signals confirm massive enrichment of SC35 signals in class 1 reflecting the IC compartment. n = number of analysed nuclei; error bars = standard deviation

Mutant and wild-type PrP immunostaining in GSS P102 L.

<p>IHC with the 3F4 antibody shows PrP deposition under diffuse, synaptic-like, and plaques aggregates in the frontal cortex (A, B) and in the cerebellar cortex (C) of case # 3; IHC with ICSM 35 in adjacent sections of the same subject immunodecorates PrP plaques in both frontal cortex (D, E) and cerebellum (F), but not diffuse or synaptic-like PrP deposits. (G) Frontal cortex from case # 3 showing 3F4-positive plaques, as well as of granular/synaptic-like PrP deposits; the punctuate and granular patterns are less evident with the ICSM 35 antibody (H), whereas a similar stain is obtained for plaques. (I) 3F4 immunostaining of the frontal cortex from case # 8 showing a widespread PrP deposition involving all cortical layers; the PrP pattern of deposition is “curly-like”; (J) immunostaining with ICSM 35 in an adjacent section shows a curly PrP deposition in the outer layer of cortex as well as a pericellular staining.</p

Co-expression of collagen type I and CD34 antigen on CD34+ co-cultured cells.

<p>Immunofluorescence analysis of collagen type I on human CD34+ cells grown in co-culture with human primary osteoblasts. Cells were labelled with anti-collagen type I antibody (red fluorescence) and with anti-human CD34 antibody (green fluorescence); nuclear counterstaining was performed with DAPI (blue fluorescence). Co-expression of collagen type I and CD34 antigen was assessed, evaluating the presence of green and red fluorescence in the same cell (D).</p

Assay IDs for quantitative real-time (QRT) PCR.

<p>Assay IDs for quantitative real-time (QRT) PCR.</p