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3 protocols using anti ferritin

1

PrPC-specific antibody characterization

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PrPC-specific antibodies 3F4 and 8H4 were obtained from Signet laboratories (Dedham, MA, USA). 3F4 binds to an epitope on amino acids 109–112 (Human PrP sequence), while 8H4 binds an epitope in the C-terminal amino acids 145–180 of human and mouse PrP62 (link). Other primary antibodies were obtained from the following sources: anti-ferritin (F5012, Sigma Aldrich, USA), anti-DMT-1 (ab55812, Abcam, USA), anti-TfR (ab84036, Abcam, USA), anti-Tf (GTX21223, GeneTex, USA), and anti-β-actin (MAB1501, Millipore, USA) Secondary antibodies conjugated with HRP were from GE Healthcare (anti-mouse, LNA931V, anti-rabbit, LNA934V). Alexa fluor-conjugated secondary antibodies were from molecular probes, ImmPACT DAB (SK4105) from Vector labs, USA, PNGase F (P0704S) from NEB, USA and Lipofectamine 3000 transfection reagent from Invitrogen, USA. Radiolabeled 59FeCl3 was obtained from Perkin-Elmer, USA. siRNA against PrP (sc36318) and scrambled siRNA (sc37007) were purchased from Santa Cruz Biotechnology Inc, USA. For immunohistochemistry of hamster retinal sections PrP-6C2 (CVI-WUR, Lelystad, Netherland), ferritin (Jackson ImmunoResearch, West Grove, PA, USA), and Iba1 (019–19741, Wako Chemicals, USA) antibodies were used. FAC (F5879) and all other chemicals were from Sigma Aldrich, USA.
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Antibodies for Protein Analysis

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The following antibodies were used in this study: Anti-PrP antibodies 3F4 and 8H4 (Signet Laboratories, Dedham, MA), and SAF32 (189720, Cayman chemicals, USA), anti-ferritin (F5012, Sigma Aldrich, USA), anti-TfR (136800, Invitrogen, USA), anti-Tf (GTX2123, GeneTEX, USA), anti-ceruloplasmin (Dako, USA), Mouse IgG (5415, Cell signaling Technology, USA), anti-Fpn (NBP1-21502, Novus biological, USA), anti β-actin (MAB1501, Milipore, USA) and anti GAPDH (GT239, GeneTEX). HRP-conjugated anti-mouse, NA931V and anti-rabbit, NA934V (GE Healthcare, UK). Alexa Fluor-conjugated secondary antibodies (molecular probes), PNGase F (P0704S, NEB, USA). NAP (Non-animal protein) blocking solution (786-190T, G-Biosciences, USA), Hoechst (#33342, Invitrogen, USA), Fluoromount-G (Southern Biotech, USA).
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3

Antibodies for Protein Analysis

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The following antibodies were used in this study: Anti-PrP antibodies 3F4 and 8H4 (Signet Laboratories, Dedham, MA), and SAF32 (189720, Cayman chemicals, USA), anti-ferritin (F5012, Sigma Aldrich, USA), anti-TfR (136800, Invitrogen, USA), anti-Tf (GTX2123, GeneTEX, USA), anti-ceruloplasmin (Dako, USA), Mouse IgG (5415, Cell signaling Technology, USA), anti-Fpn (NBP1-21502, Novus biological, USA), anti β-actin (MAB1501, Milipore, USA) and anti GAPDH (GT239, GeneTEX). HRP-conjugated anti-mouse, NA931V and anti-rabbit, NA934V (GE Healthcare, UK). Alexa Fluor-conjugated secondary antibodies (molecular probes), PNGase F (P0704S, NEB, USA). NAP (Non-animal protein) blocking solution (786-190T, G-Biosciences, USA), Hoechst (#33342, Invitrogen, USA), Fluoromount-G (Southern Biotech, USA).
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