A control, the brain homogenate from sCJD directly loaded onto the

A control, the brain homogenate from sCJD directly loaded onto the gel was probed with 3F4. (TIF)Preparation of brain homogenate, S2, and P2 fractionsThe 10 (w/v) brain homogenates were prepared in 9 volumes of lysis buffer (10 mM Tris, 150 mM NaCl, 0.5 Nonidet P-40, 0.5 deoxycholate, 5 mM EDTA, pH 7.4) with pestle on ice. When required, brain homogenates were K162 centrifuged at 1,000 g for 10 min at 4uC. In order to prepare S2 and P2 fractions, the supernatants (S1) were further centrifuged at 35,000 rpm (100,000 g) for 1 hour at 4uC. After the ultracentrifugation, the detergentsoluble fraction was recovered in the supernatants (S2) while the detergent-insoluble fraction (P2) was recovered in the pellets that were resuspended in lysis buffer as described [18].Specific capture of PrPSc by gene 5 protein and immunoprecipitation of PrP by 6H4 antibodyThe preparation of gene 5 protein (g5p) conjugated magnetic beads and specific capture of PrPSc by g5p beads were conducted as previously described [18]. The immunoprecipitation of PrP from brain homogenates and cell lysates by 6H4-conjugated magnetic beads was performed as previously described [16,18].ImmunoblottingSamples treated with or without PK-digestion were resolved on 15 Tris-HCl Criterion (Bio-Rad) as described previously [18]. The following anti-PrP antibodies were used (Fig. S1): mouse monoclonal antibody (mAb) 3F4 directed against human PrP residues 106?10 [38,39], 1E4 against human PrP residues 97?105 [10,18], 6H4 against human PrP144?52 (Prionics AG, Switzerland), V14 recognizing the human PrP185?96 [11-13], Pc248 directed against the octarepeat region [12,40], and Bar209 anti-mono197 and unglycosylated PrP mAb [12] (Fig. S1), recognizing a conformational epitope [12] likely involving human PrP168-181, like anti-mono197 PrP mAb V61 [12].AcknowledgmentsThe authors want to thank Janis Blevins and Jeffrey Negrey for coordinating brain tissues and clinical information, Jacques Grassi and Stephanie Simon for SMER28 supplier providing the Bar209 antibody, Dr. Martin Hermann ?Grouschup for providing brains of Tg mice expressing PrP glycosylation mutants, Drs. Geoff Kneale and John McGeehan for providing g5p, as well as Dr. Janet Byron Anderson and Paul Curtiss for proofreading the manuscript. Y.A.Z. was supported by a grant from the Chinese National Key Clinical Department Project.Immunofluorescence and confocal microscopyImmunofluorescence staining of PrP in transfected cells expressing PrPWt, PrPV180I, or PrPT183A was performed as previously described [11]. In brief, the cells were fixed in 4 paraformaldehyde. After blocking with PBST (10 Goat Serum, 2 T-20, 1 Triton X-100), the cells were then incubated with 3F4 (1:10,000) or calnexin (1:1,000) at room temperature, rinsed with PBS, followed with FITC-conjugated goat anti-mouse IgG at 1:1,000 (Sigma, St. Louis, MO) or Alexa Fluor 568-conjugated goat anti-rabbit as secondary antibody. Microscopy was performed using a high-speed Leica SP5 Broadband confocal microscope (Wetzlar, Germany) and HCX PL APO CS 636 oil immersion objective (NA 1.4). Images were acquired and analyzed using Leica Application Suite software. Further analysis for colocalization and correlation was performed using Imaris imaging suite (Bitplane, St. Paul, MN).Author ContributionsInitiated and coordinated the entire project: WQZ. Revised the manuscript: XX SH IC MM JL QK JPB BAC RBP. Conceived and designed the experiments: MM RBP WQZ. Performed the experiments: XX JY SH IC YAZ.A control, the brain homogenate from sCJD directly loaded onto the gel was probed with 3F4. (TIF)Preparation of brain homogenate, S2, and P2 fractionsThe 10 (w/v) brain homogenates were prepared in 9 volumes of lysis buffer (10 mM Tris, 150 mM NaCl, 0.5 Nonidet P-40, 0.5 deoxycholate, 5 mM EDTA, pH 7.4) with pestle on ice. When required, brain homogenates were centrifuged at 1,000 g for 10 min at 4uC. In order to prepare S2 and P2 fractions, the supernatants (S1) were further centrifuged at 35,000 rpm (100,000 g) for 1 hour at 4uC. After the ultracentrifugation, the detergentsoluble fraction was recovered in the supernatants (S2) while the detergent-insoluble fraction (P2) was recovered in the pellets that were resuspended in lysis buffer as described [18].Specific capture of PrPSc by gene 5 protein and immunoprecipitation of PrP by 6H4 antibodyThe preparation of gene 5 protein (g5p) conjugated magnetic beads and specific capture of PrPSc by g5p beads were conducted as previously described [18]. The immunoprecipitation of PrP from brain homogenates and cell lysates by 6H4-conjugated magnetic beads was performed as previously described [16,18].ImmunoblottingSamples treated with or without PK-digestion were resolved on 15 Tris-HCl Criterion (Bio-Rad) as described previously [18]. The following anti-PrP antibodies were used (Fig. S1): mouse monoclonal antibody (mAb) 3F4 directed against human PrP residues 106?10 [38,39], 1E4 against human PrP residues 97?105 [10,18], 6H4 against human PrP144?52 (Prionics AG, Switzerland), V14 recognizing the human PrP185?96 [11-13], Pc248 directed against the octarepeat region [12,40], and Bar209 anti-mono197 and unglycosylated PrP mAb [12] (Fig. S1), recognizing a conformational epitope [12] likely involving human PrP168-181, like anti-mono197 PrP mAb V61 [12].AcknowledgmentsThe authors want to thank Janis Blevins and Jeffrey Negrey for coordinating brain tissues and clinical information, Jacques Grassi and Stephanie Simon for providing the Bar209 antibody, Dr. Martin Hermann ?Grouschup for providing brains of Tg mice expressing PrP glycosylation mutants, Drs. Geoff Kneale and John McGeehan for providing g5p, as well as Dr. Janet Byron Anderson and Paul Curtiss for proofreading the manuscript. Y.A.Z. was supported by a grant from the Chinese National Key Clinical Department Project.Immunofluorescence and confocal microscopyImmunofluorescence staining of PrP in transfected cells expressing PrPWt, PrPV180I, or PrPT183A was performed as previously described [11]. In brief, the cells were fixed in 4 paraformaldehyde. After blocking with PBST (10 Goat Serum, 2 T-20, 1 Triton X-100), the cells were then incubated with 3F4 (1:10,000) or calnexin (1:1,000) at room temperature, rinsed with PBS, followed with FITC-conjugated goat anti-mouse IgG at 1:1,000 (Sigma, St. Louis, MO) or Alexa Fluor 568-conjugated goat anti-rabbit as secondary antibody. Microscopy was performed using a high-speed Leica SP5 Broadband confocal microscope (Wetzlar, Germany) and HCX PL APO CS 636 oil immersion objective (NA 1.4). Images were acquired and analyzed using Leica Application Suite software. Further analysis for colocalization and correlation was performed using Imaris imaging suite (Bitplane, St. Paul, MN).Author ContributionsInitiated and coordinated the entire project: WQZ. Revised the manuscript: XX SH IC MM JL QK JPB BAC RBP. Conceived and designed the experiments: MM RBP WQZ. Performed the experiments: XX JY SH IC YAZ.

Leave a Reply