Journal: PLoS ONE

Article Title: Investigating CRISPR/Cas9 gene drive for production of disease-preventing prion gene alleles

doi: 10.1371/journal.pone.0269342

Figure Lengend Snippet: (A) Sanger sequencing chromatograms for mice derived from fertilized FVB/NJ oocytes that were electroporated with recCas9/ Prnp gRNA–3 RNP complexes. The numbers below the automated base calls indicate the position in the sequencing read rather than the Prnp ORF. Changes to the PrP C amino acid sequence for the lines with disrupted Prnp are indicated in the bottom box. (B) Agarose gel image showing the products of T7E1 mismatch cleavage assay reactions for negative (homoduplex) and positive (heteroduplex) control DNA solutions and DNA obtained from the founder of line 34. The presence of the bands indicated by the red arrows confirms that line 34 has a disrupted Prnp allele. (C) Capillary western images confirming that PrP C expression is undetectable in brain homogenates from homozygous line 33 mice. Sha31 and 12B2 are two different PrP C antibodies. New abbreviation: KO, knockout.

Article Snippet: Primary antibodies were Sha31 anti-PrP (Spi-Bio Inc., A03213; diluted 1:10000), 12B2 anti-PrP (see Ref. [ ]; diluted 1:500) and anti-beta-tubulin (Novus Biologicals, NB600-936; diluted 1:200).

Techniques: Sequencing, Derivative Assay, Agarose Gel Electrophoresis, Cleavage Assay, Control, Western Blot, Expressing, Knock-Out

Journal: Prion

Article Title: Cellular prion protein distribution in the vomeronasal organ, parotid, and scent glands of white-tailed deer and mule deer

doi: 10.1080/19336896.2022.2079888

Figure Lengend Snippet: Relative pixel intensity analysis of PrP C expression in facial integumentary clarified 10% (w/v) gland homogenates of mule deer (A, C, E) and white-tailed deer (B, D, F). Background-adjusted average pixel intensities of PrP C bands of forehead (a-b), preorbital (c-d), and vestibular nasal glands were compared between lanes of SDS-PAGE PVDF membranes probed with anti-PrP Sha31. Unclarified white-tailed deer whole brain homogenate was used for reference. Sample size, mean, 95% confidence intervals, and significance by Mann-Whitney tests are shown.

Article Snippet: PrP C was probed with the mouse IgG 1 anti-prion protein SHA31 monoclonal primary antibody (1:10,000) (Cayman Chemical, USA) which binds to an epitope of 148–155 ( Odocoileus amino acid sequence).

Techniques: Expressing, SDS Page, MANN-WHITNEY

Journal: Prion

Article Title: Cellular prion protein distribution in the vomeronasal organ, parotid, and scent glands of white-tailed deer and mule deer

doi: 10.1080/19336896.2022.2079888

Figure Lengend Snippet: Protein concentration-adjusted PrP C protein expression in deer exocrine glands. Capillary electrophoresis immunoassay chemiluminescence sample size, mean, and 95% confidence intervals of clarified 10% (w/v) deer gland homogenates prepared in RIPA buffer. Deer facial (a-e) and leg (f-h) tissue and gland homogenate samples were adjusted to final protein concentrations of 1.5 μg/μL for the immunoassay. PrP C signal was detected by anti-PrP SHA31 antibody.

Article Snippet: PrP C was probed with the mouse IgG 1 anti-prion protein SHA31 monoclonal primary antibody (1:10,000) (Cayman Chemical, USA) which binds to an epitope of 148–155 ( Odocoileus amino acid sequence).

Techniques: Protein Concentration, Expressing, Electrophoresis

Journal: Prion

Article Title: Cellular prion protein distribution in the vomeronasal organ, parotid, and scent glands of white-tailed deer and mule deer

doi: 10.1080/19336896.2022.2079888

Figure Lengend Snippet: Species and sex influence on PrP C detection by anti-PrP SHA31 capillary gel electrophoresis assay with total protein concentration-standardized 10% (w/v) clarified gland homogenates

Article Snippet: PrP C was probed with the mouse IgG 1 anti-prion protein SHA31 monoclonal primary antibody (1:10,000) (Cayman Chemical, USA) which binds to an epitope of 148–155 ( Odocoileus amino acid sequence).

Techniques: Nucleic Acid Electrophoresis, Concentration Assay

Journal: Prion

Article Title: Cellular prion protein distribution in the vomeronasal organ, parotid, and scent glands of white-tailed deer and mule deer

doi: 10.1080/19336896.2022.2079888

Figure Lengend Snippet: Quantified PrP C protein concentration in deer gland homogenates. Total PrP C concentrations of individuals, means, and 95% confidence intervals of clarified 10% (w/v) mule deer (MD) and white-tailed deer (WT) facial (a-e) and leg (f-h) gland homogenates prepared in RIPA buffer as determined by SHA31-N5 sandwich ELISA. Protein concentration was calculated using a full-length recombinant deer prion protein standard curve.

Article Snippet: PrP C was probed with the mouse IgG 1 anti-prion protein SHA31 monoclonal primary antibody (1:10,000) (Cayman Chemical, USA) which binds to an epitope of 148–155 ( Odocoileus amino acid sequence).

Techniques: Protein Concentration, Sandwich ELISA, Recombinant

Journal: Prion

Article Title: Cellular prion protein distribution in the vomeronasal organ, parotid, and scent glands of white-tailed deer and mule deer

doi: 10.1080/19336896.2022.2079888

Figure Lengend Snippet: Mean PrP C concentrations and 95% confidence intervals of clarified 10% (w/v) gland homogenates as determined by SHA31-N5 sandwich ELISA

Article Snippet: PrP C was probed with the mouse IgG 1 anti-prion protein SHA31 monoclonal primary antibody (1:10,000) (Cayman Chemical, USA) which binds to an epitope of 148–155 ( Odocoileus amino acid sequence).

Techniques: Concentration Assay

Journal: Acta neuropathologica

Article Title: Characterization of prion strains and peripheral prion infectivity patterns in E200K genetic CJD patients.

doi: 10.1007/s00401-025-02903-5

Figure Lengend Snippet: Fig. 1 PrPres western blot profiles in original E200K gCJD isolates and E200K gCJD-inoc- ulated human PrP-expressing mice. Western blot analysis of proteinase K-resistant prion protein (PrPres) profiles was performed on brain homogen- ates from both the original E200K gCJD patient isolates and transgenic mice expressing human PrP with Met129 (tgMet) or Val129 (tgVal) at codon 129. Mice were intracerebrally inoculated with 20 µL of 10% brain homogenate from E200K gCJD patients (n = 6 per group). Two serial passages were carried out in each mouse line (details in Table 1). The PrPres isoform (type 1 or type 2) was identified via SDS-PAGE and Western blot using the anti-PrP monoclonal antibody Sha31 (epitope YEDRYYRE). To control for PrPres isoform, MM1 sCJD (type 1) and VV2 sCJD (type 2) isolates were included in each gel. The PrPres isoform results for each passage and mouse line are summarized in Table 1

Article Snippet: Immunodetection was carried on PVDF membranes out using the monoclonal primary antibody Sha31 (1 μg/mL), which recognizes amino acids 145–152 (YEDRYYRE) of PrP, and an anti-mouse HRP-conjugated secondary antibody (Biorad) [16].

Techniques: Western Blot, Expressing, Transgenic Assay, SDS Page, Control

Journal: Acta neuropathologica

Article Title: Characterization of prion strains and peripheral prion infectivity patterns in E200K genetic CJD patients.

doi: 10.1007/s00401-025-02903-5

Figure Lengend Snippet: Fig. 3 PrPres western blot in the brains of tgMet mice inoculated with brain and peripheral tissues from E200K gCJD Patients. Western blot analysis of proteinase K-resistant prion protein (PrPres) was conducted on brain homogenates from transgenic mice expressing human PrP with Met129 (tgMet) after two serial intracerebral passages (n = 6 per group). The mice were inoculated with 20µL of a 10% homogenate prepared from brain or peripheral tissues of E200K gCJD patients (refer to Tables 1 and 2). PrPres was detected using the anti-PrP mon- oclonal antibody Sha31, targeting the YEDRYYRE epitope. For com- parison, MM1 sCJD (type 1) and VV2 sCJD (type 2) prion isolates were included as controls. PrPres isoform identification results for each passage are summarized in Table 2

Article Snippet: Immunodetection was carried on PVDF membranes out using the monoclonal primary antibody Sha31 (1 μg/mL), which recognizes amino acids 145–152 (YEDRYYRE) of PrP, and an anti-mouse HRP-conjugated secondary antibody (Biorad) [16].

Techniques: Western Blot, Transgenic Assay, Expressing

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: The Scrapie Prevalence in a Goat Herd Is Underestimated by Using a Rapid Diagnostic Test

doi: 10.3389/fbioe.2020.00164

Figure Lengend Snippet: Western blot profile of digested brain samples from selected goats using two monoclonal antibodies. (A) Sha31 antibody. (B) P4 antibody. Lanes 1 and 10: molecular mass marker; lane 2: goat 2135 (clinical suspect, positive on brain by IHC and ELISA); lane 3: goat 2113, clinical suspect, positive on brain by IHC and ELISA); lane 4: goat 2078 (no clinical signs of scrapie, positive on brain by IHC, negative by ELISA); lane 5: goat 2117 (no clinical signs of scrapie, negative on brain by IHC and ELISA, positive on lymphoid tissue by IHC; scrapie profile difficult to discern with the picture contrast used); lane 6: goat 2102 (clinical suspect, negative on brain by ELISA and on all tissues by IHC); lane 7: caprine classical scrapie control (RSCRAP 17/00006, II 142 QQ 222 ); lane 8: bovine classical BSE control (RBSE 98/00291); lane 9: ovine classical scrapie control (PG1903/97).

Article Snippet: Primary antibodies were Sha31 (Bio-Rad Laboratories, directed against the 148-YEDRYYRE-155 epitope), which was prepared according to the manufacturer’s kit instructions, and the N-terminal antibody P4 (R-Biopharm Rhône Ltd., Glasgow, United Kingdom, directed against the 93-WGQGGSH-99 epitope), which was used in a dilution 1:5000 of a stock solution of 1 mg/ml.

Techniques: Western Blot, Marker, Enzyme-linked Immunosorbent Assay

Journal: Veterinary Research

Article Title: L-BSE experimentally transmitted to sheep presents as a unique disease phenotype

doi: 10.1186/s13567-016-0394-1

Figure Lengend Snippet: WB images from primary passage samples. A and B Nine representative recipients, homozygous for alanine (codon 136) detected by Sha31 ( A ), or P4 ( B ). (Lane 1, 63/11; lane 2, 3/11; lane 3, 457/11; lane 4 26/12; lane 5 112/12; lane 6, 182/12; lane 7, 3/13; lane 8, 98/11; lane 9, 455/11; AB + , donor bovine L-BSE; B + , bovine C-BSE; S + , ovine Scrapie; BS + , experimental ovine CBSE; S-, negative sheep; M, molecular mass markers. A 1 min exposure, B 10 min exposure). C and D Eight representative recipients homozygous or heterozygous for valine (codon 136), detected by Sha31 ( C ) or P4 ( D ), (Lane 1, case 1591/10; lane 2, case 58/11; lane 3, 140/11; lane 4, 267/11; lane 5, 456/11; lane 6, 113/12; lane 7, 4/12; lane 8, 167/12; markers and controls as for A and B . C 1 min exposure, D 10 min exposure). There is low molecular mass migration of the unglycosylated band (arrow), similar to that of the donor bovine L-BSE (AB+), for all but one of the ovine recipients (VRQ/VRQ Lane 8 C , D ) regardless of genotype. Similar di-;mono-glycosylated band ratios are also seen in all cases when detected by mAb Sha31 (see Additional file ). All recipient samples, irrespective of genotype, are also detected with mAb P4 ( B , D ). In contrast the donor L-BSE case (AB+) is not. Following extraction with a Proteinase K digestion step, may contain a mixture of varying molecular mass fragments, partly due to multiple cleavage sites and variability in resistance of PrP Sc to the concentration of the enzyme. The two additional lower bands observed in these profiles are regularly observed in diagnostic samples processed in this way. For diagnostic analysis they are disregarded. Only the standard three bands pertaining to the di, mono and un-glycosylated forms of PrP Sc are considered relevant.

Article Snippet: The primary antibodies were Sha31 (prepared according to the kit protocol) and P4 (0.2 µg/mL, RIDA ® , r-Biopharm).

Techniques: Migration, Concentration Assay, Diagnostic Assay

Journal: Veterinary Research

Article Title: L-BSE experimentally transmitted to sheep presents as a unique disease phenotype

doi: 10.1186/s13567-016-0394-1

Figure Lengend Snippet: WB for ARQ/VRQ subpassage recipients. A Sha31 and P4 blots of the ARQ/VRQ donor and its ARQ/VRQ recipients. (Lane 1, case 1591/10 (ARQ/VRQ Donor), Lane 2, case 6/14; Lane 3, case 73/14; Lane 4, case 76/14 Lane 5, case 78/14; Lane 6, case 79/14 M, Molecular marker; B+, Bovine BSE; S+, Classical Ovine Scrapie. This panel comprises a mixture of 1 and 10 min exposures). B Sha31 and P4 blots of the AFRQ/AFRQ donor and its ARQ/VRQ recipients (Lanes 1 and 2, case 98/11 (AFRQ/AFRQ Donor); Lane 3, case 80/14; Lane 4, case 81/14; Lane 5, case 82/14; Lane 6, case 75/14; M, molecular marker; S+, classical ovine scrapie; B+, bovine BSE. This panel comprises a mixture of 1 and 10 min exposures). In contrast the donor L-BSE case (AB+)molecular characteristics described for primary passage (Figure ; Additional files A–E) are retained on subpassage for all recipient animals regardless of the donor.

Article Snippet: The primary antibodies were Sha31 (prepared according to the kit protocol) and P4 (0.2 µg/mL, RIDA ® , r-Biopharm).

Techniques: Marker

Journal: The Journal of Biological Chemistry

Article Title: Chronic wasting disease (CWD) prion strains evolve via adaptive diversification of conformers in hosts expressing prion protein polymorphisms

doi: 10.1074/jbc.RA120.012546

Figure Lengend Snippet: Novel PrP CWD properties following Wisc-1 propagation in deer expressing PrP C polymorphisms. PrP CWD profiles were obtained from whole hemi-encephalon homogenates derived from orally-infected white-tailed deer expressing different PrP C primary structures ( PRNP alleles). A, PK-resistant (res) PrP CWD signatures detected with monoclonal antibodies (mAbs) 12B2 (deer; 93–97), 8G8 (100–105), BAR224 (146–156), and 8H4 (179–189). Expression of histidine or serine at residues 95 and 96, respectively, disrupted 12B2 detection of these PrP CWD allelotypes. For other mAbs, detection of His-95/Ser-96 PK-res PrP CWD required three times more brain protein equivalents. UI, uninfected control. Previously described detection with 8G8 mAb was included for the purpose of comparison. B, linear representation and partial primary sequence of deer PrP C displaying amino acid polymorphisms ( red ), secondary structural motifs ( white boxes : β, β-sheets; α, α-helices), and the epitopes of mAb used for detection; Sha31(148–155). Residues 184 and 200 indicate variable N -linked glycosylation residues; GLP , glycolipid.

Article Snippet: Detection was performed using primary monoclonal antibodies: BAR224 (0.2 μg/ml diluted 1:10,000 in 5% (w/v) nonfat dry milk in TBST; Cayman Chemical); 8G8 (0.2 μg/ml diluted 1:2000; Cayman Chemical); 12B2 (0.2 μg/ml diluted 1:20,000 in TBST; Wageningen UR); 8H4 (0.2 μg/ml diluted 1:10,000; Abcam); Sha31 (0.2 μg/ml diluted: 30,000 in TBST; Cayman Chemical).

Techniques: Expressing, Derivative Assay, Infection, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Chronic wasting disease (CWD) prion strains evolve via adaptive diversification of conformers in hosts expressing prion protein polymorphisms

doi: 10.1074/jbc.RA120.012546

Figure Lengend Snippet: Alternative N-terminal endoproteolytic processing and antibody reactivity of total PrP in brain of CWD-infected deer expressing different PrP C allelotypes. A, de-glycosylated total PrP in Wisc-1–infected white-tailed deer expressing WT-(Q95G96)–PrP C , Ser-96–PrP C , or His-95–PrP C allelotypes. His-95/Ser-96 deer accumulated a distinctive N-terminally–cleaved PrP ( C3 ) of ∼20 kDa following removal of glycans with PNGase F. Full-length ( FL ) PrP, C-terminal PrP ( C1, C2, and C3 ). B, percentage of de-glycosylated C3- and C2–PrP detected with BAR224. Mean with standard deviation is indicated by bars . Densitometry was performed in three separate de-glycosylation experiments for each individual deer CWD lineage. The brain of His-95/Ser-96 deer contained significantly higher levels of C3- and C2–PrP than other deer. ANOVA, p < 0.05. Differences in C2–PrP abundance were 4–5-fold larger with 8G8 (data not shown). C , total deer brain PrP ( G , glycosylated; UG, unglycosylated). The mAb 12B2 (epitope WGQGG, deer residues 93–97) does not recognize Ser-96–PrP C or His-95–PrP C . The abundance of full-length and C1-PrP between deer was equivalent ruling out post-homogenization degradation.

Article Snippet: Detection was performed using primary monoclonal antibodies: BAR224 (0.2 μg/ml diluted 1:10,000 in 5% (w/v) nonfat dry milk in TBST; Cayman Chemical); 8G8 (0.2 μg/ml diluted 1:2000; Cayman Chemical); 12B2 (0.2 μg/ml diluted 1:20,000 in TBST; Wageningen UR); 8H4 (0.2 μg/ml diluted 1:10,000; Abcam); Sha31 (0.2 μg/ml diluted: 30,000 in TBST; Cayman Chemical).

Techniques: Infection, Expressing, Standard Deviation, Homogenization

Journal: The Journal of Biological Chemistry

Article Title: Chronic wasting disease (CWD) prion strains evolve via adaptive diversification of conformers in hosts expressing prion protein polymorphisms

doi: 10.1074/jbc.RA120.012546

Figure Lengend Snippet: The CDI measurements of WT–PrP were performed with Eu-12B2 mAb and PrP with His-95 or Ser-96 polymorphisms with Eu-8G8 mAb

Article Snippet: Detection was performed using primary monoclonal antibodies: BAR224 (0.2 μg/ml diluted 1:10,000 in 5% (w/v) nonfat dry milk in TBST; Cayman Chemical); 8G8 (0.2 μg/ml diluted 1:2000; Cayman Chemical); 12B2 (0.2 μg/ml diluted 1:20,000 in TBST; Wageningen UR); 8H4 (0.2 μg/ml diluted 1:10,000; Abcam); Sha31 (0.2 μg/ml diluted: 30,000 in TBST; Cayman Chemical).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Chronic wasting disease (CWD) prion strains evolve via adaptive diversification of conformers in hosts expressing prion protein polymorphisms

doi: 10.1074/jbc.RA120.012546

Figure Lengend Snippet: Conformational diversification of Wisc-1 prions in deer expressing PrP C polymorphisms Ser-96 and His-95. Structural stability of deer PrP CWD after exposure to increasing concentrations of GdnHCl ( m ). The apparent fractional change ( F app ) of unfolding was measured by CDI with 8G8 or 12B2 mAbs before or after proteinase K treatment ( PK −/+). CWD conformers encoded in all three PrP primary structures were detected with 8G8 mAb, whereas 12B2 only recognized prions encoded on WT–PrP. Black dotted lines indicate the denaturation midpoints (GdnHCl 1/2 ). F app values and bars represent the mean ± S.E. obtained for each individual deer CWD lineage (two batches of 20% w/v brain homogenate) measured in triplicate with each Eu-labeled mAb.

Article Snippet: Detection was performed using primary monoclonal antibodies: BAR224 (0.2 μg/ml diluted 1:10,000 in 5% (w/v) nonfat dry milk in TBST; Cayman Chemical); 8G8 (0.2 μg/ml diluted 1:2000; Cayman Chemical); 12B2 (0.2 μg/ml diluted 1:20,000 in TBST; Wageningen UR); 8H4 (0.2 μg/ml diluted 1:10,000; Abcam); Sha31 (0.2 μg/ml diluted: 30,000 in TBST; Cayman Chemical).

Techniques: Expressing, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Chronic wasting disease (CWD) prion strains evolve via adaptive diversification of conformers in hosts expressing prion protein polymorphisms

doi: 10.1074/jbc.RA120.012546

Figure Lengend Snippet: Sedimentation properties and protease resistance of PrP CWD from Wisc-1 deer CWD lineages. Fractions collected from the bottom of the tube were analyzed by Western blotting and CDI prior (−) or after (+) treatment with proteinase K. A, distribution of total and PK-res WT–PrP CWD in different CWD allotypes detected with 12B2 mAb. B, concentration of fractionated WT–PrP C ( green ), total WT–PrP CWD ( orange ), and PK-res WT–PrP CWD ( black ) was determined for WT/WT, Ser-96/WT, and His-95/WT prions using CDI with 12B2 mAb. Detection of His-95 and Ser-96 PrP CWD required 8G8 mAb. Bars represent average ± S.E. CDI measurements were performed on each individual deer sample in triplicate.

Article Snippet: Detection was performed using primary monoclonal antibodies: BAR224 (0.2 μg/ml diluted 1:10,000 in 5% (w/v) nonfat dry milk in TBST; Cayman Chemical); 8G8 (0.2 μg/ml diluted 1:2000; Cayman Chemical); 12B2 (0.2 μg/ml diluted 1:20,000 in TBST; Wageningen UR); 8H4 (0.2 μg/ml diluted 1:10,000; Abcam); Sha31 (0.2 μg/ml diluted: 30,000 in TBST; Cayman Chemical).

Techniques: Sedimentation, Western Blot, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Chronic wasting disease (CWD) prion strains evolve via adaptive diversification of conformers in hosts expressing prion protein polymorphisms

doi: 10.1074/jbc.RA120.012546

Figure Lengend Snippet: Co-existence of Wisc-1 and H95 + conformers encoded in WT–PrP from tg33 mice. Structural properties of WT–PrP CWD in tg33 mice were exposed to Wisc-1 CWD (WT/WT) or the His-95/Ser-96 (a mixture of Wisc-1 and emergent H95 + strains) are shown. A, sedimentation velocity profiles. The concentration of fractionated WT–PrP C ( green ), total WT–PrP CWD ( orange ), and PK-res PrP CWD ( black ) was determined using CDI with 8G8 mAb. Bars represent mean ± S.E. measured for 3–4 mice by triplicate. B, average structural stability ( yellow ) of total PrP CWD (PK−) measured for groups of tg33 mice. Brain homogenates from tg33 mice contained PrP CWD conformers with distinct structural stability separated by sedimentation velocity ( bar 7, red, and bar 2, blue ). The apparent fractional change ( F app ) of unfolding was determined by CDI using 8G8 mAb. Black dotted lines indicate the denaturation midpoints (GdnHCl 1/2 m ). Bars represent mean ± S.E. Sedimentation and conformation analysis were performed in 3–5 mice per passage line.

Article Snippet: Detection was performed using primary monoclonal antibodies: BAR224 (0.2 μg/ml diluted 1:10,000 in 5% (w/v) nonfat dry milk in TBST; Cayman Chemical); 8G8 (0.2 μg/ml diluted 1:2000; Cayman Chemical); 12B2 (0.2 μg/ml diluted 1:20,000 in TBST; Wageningen UR); 8H4 (0.2 μg/ml diluted 1:10,000; Abcam); Sha31 (0.2 μg/ml diluted: 30,000 in TBST; Cayman Chemical).

Techniques: Sedimentation, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Chronic wasting disease (CWD) prion strains evolve via adaptive diversification of conformers in hosts expressing prion protein polymorphisms

doi: 10.1074/jbc.RA120.012546

Figure Lengend Snippet: Selection of strain-specific prion conformers in transgenic mice expressing deer PrP C polymorphisms. Conformational stability of different CWD lineages propagated ( i.e. encoded) in Gly-96 (WT) or Ser-96–PrP from transgenic mice. P0 refers to the unfolding of Wisc-1 replication products after homologous prion conversion in WT/WT deer and heterologous conversion in Ser-96/WT, His-95/WT, and His-95/Ser-96 deer. P1 describes the GdnHCl unfolding resistance of PrP CWD conformational descendents stably amplified after first passages in tg33 (WT; yellow ) and tg60 (Ser-96; purple ) mice. The apparent fractional change ( F app ) of unfolding was determined by CDI with 8G8 mAb after proteinase K treatment. Black dotted lines indicate the denaturation midpoints (GdnHCl 1/2 ). The H95 + strain conformers differed in their adaptability in mice expressing serine instead of glycine at amino acid 96. PK-res PrP CWD was either absent or below detection levels in WT/WT and Ser-96/WT infected tg60 mice. F app values and bars represent the mean ± S.E. measured in 10% w/v brain homogenates from three to five prion disease affected mice and measured in triplicate with Eu-8G8-labeled mAb.

Article Snippet: Detection was performed using primary monoclonal antibodies: BAR224 (0.2 μg/ml diluted 1:10,000 in 5% (w/v) nonfat dry milk in TBST; Cayman Chemical); 8G8 (0.2 μg/ml diluted 1:2000; Cayman Chemical); 12B2 (0.2 μg/ml diluted 1:20,000 in TBST; Wageningen UR); 8H4 (0.2 μg/ml diluted 1:10,000; Abcam); Sha31 (0.2 μg/ml diluted: 30,000 in TBST; Cayman Chemical).

Techniques: Selection, Transgenic Assay, Expressing, Stable Transfection, Amplification, Infection, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Chronic wasting disease (CWD) prion strains evolve via adaptive diversification of conformers in hosts expressing prion protein polymorphisms

doi: 10.1074/jbc.RA120.012546

Figure Lengend Snippet: PrP CWD abundance in CWD-infected transgenic mice and phenotypic stability after allogenic prion passage. The average levels of PrP C , total PrP CWD , and PK-res PrP CWD were measured by CDI with 8G8 mAb. A, Tg60 mice exposed to WT/WT ( i.e. Wisc-1) and Ser-96/WT deer prions accumulated low levels of PK-sen PrP CWD compared with mice receiving His-95/WT and His-95/Ser-96 ( i.e. H95 + ) prions. B, Tg33 mice accumulated similar levels of PrP CWD . C, host replication of PK-sen S96 PrP CWD in tg33 mice ( D ) (Bar224 1:10.000). D, lesion profiles in sagittal brain sections from tg33 mice infected with tg60P1 prions. Score values represent the mean ± S.E. (two-way ANOVA; *, p < 0.05). Lesion profile of tg33P2WT/WT ( black ) is included for comparison. The lesion profile of tg33 mice infected with tg60P1His-95/Ser-96 prions ( light blue ) was not included in the statistical comparison. The H95 + strain brain region targeting was similar in tg33 and tg60 mice. Brain regions: 1, cerebral cortex; 2, cerebral nuclei; 3, hippocampus; 4, thalamus; 5, hypothalamus; 6, midbrain; 7, pons; 8, medulla; and 9. cerebellum. E, low abundance of novel S96 res types following first passage of WT/WT and Ser-96/WT CWD into individual tg60 mice in the absence of prion disease signs. Brains of tg-mice infected with Wisc-1 and H95 + ( a and b ), respectively, contained more PrP CWD than asymptomatic tg60 carriers ( c–g ) as measured by Western blotting. Wisc-1 and H95 + strains were diluted 100- and 10-fold, respectively, in normal tg60 brain homogenate prior parallel PK treatment. Unlike standard detection conditions, a range between 240 and 300 μg (3.4–4.2 μg/μl) of brain total protein equivalents was incubated with 50–100 μg/ml of PK (70 μl reaction volume) to enhance detection. Samples (≈25–31 μg of protein equivalents-post PK) were resolved per well (Sha31 mAb 1:30,000). CDI analysis was performed in 3–5 mice per passage line with the exception of asymptomatic tg60P1(two mice). Histological analysis and statistical comparison were performed in groups of three mice per passage line.

Article Snippet: Detection was performed using primary monoclonal antibodies: BAR224 (0.2 μg/ml diluted 1:10,000 in 5% (w/v) nonfat dry milk in TBST; Cayman Chemical); 8G8 (0.2 μg/ml diluted 1:2000; Cayman Chemical); 12B2 (0.2 μg/ml diluted 1:20,000 in TBST; Wageningen UR); 8H4 (0.2 μg/ml diluted 1:10,000; Abcam); Sha31 (0.2 μg/ml diluted: 30,000 in TBST; Cayman Chemical).

Techniques: Infection, Transgenic Assay, Western Blot, Incubation