Mitochondrial and Autophagy Protein Detection

Mitochondrial primary antibodies: TOMM20 (Santa Cruz), TIMM9 (Abcam), NDUFA9 (Abcam), COX4i1 (Abcam), ATP5A (Abcam), β-ACTIN (Sigma-Aldrich). Horseradish peroxidase (HRP)-linked secondary antibodies were used (Sigma-Aldrich). LC3 primary antibodies: Rabbit anti-LC3 (Novus Biologicals; NB100–2220) used at 1:1000 dilution; mouse anti-actin (Sigma A5316) used at 1:500 dilution. Secondary antibodies: Polyclonal goat anti-rabbit immunoglobulins/HRP (Dako P0448) used at 1:5000; polyclonal goat anti-mouse immunoglobulins/HRP (P0447) used at 1:5000. Beta- and Gamma-synuclein primary antibodies: mAb α/β-Synuclein (Syn205, Cell Signaling; 1:1000) or pAb γ1-Synuclein (1:1000). γ1-Synuclein polyclonal antibody was raised to the peptide DFSHGGMEGGEGGEGY by immunization of rabbits (New England Peptide) and affinity purified as previously described (54 (link)). IRDye-800 and IRDye-680 (LI-COR, Lincoln, NE) conjugated secondary antibodies (1:10 000) enabled the blot to be imaged using an Odyssey Infrared Imager (catalog no. 9120; LI-COR) with a wide linear range.

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Keatinge M., Bui H., Menke A., Chen Y.C., Sokol A.M., Bai Q., Ellett F., Da Costa M., Burke D., Gegg M., Trollope L., Payne T., McTighe A., Mortiboys H., de Jager S., Nuthall H., Kuo M.S., Fleming A., Schapira A.H., Renshaw S.A., Highley J.R., Chacinska A., Panula P., Burton E.A., O'Neill M.J, & Bandmann O. (2015). Glucocerebrosidase 1 deficient Danio rerio mirror key pathological aspects of human Gaucher disease and provide evidence of early microglial activation preceding alpha-synuclein-independent neuronal cell death. Human Molecular Genetics, 24(23), 6640-6652.

Publication 2015

TOMM20 TIMM9 NDUFA9 COX4i1 ATP5A β-ACTIN mouse anti-actin Polyclonal goat anti-rabbit immunoglobulins/HRP polyclonal goat anti-mouse immunoglobulins/HRP IRDye-800 IRDye-680 Odyssey Infrared Imager

Actin Anti immunoglobulins Antibodies Antibody Biologicals Dilution Gamma synuclein Goat Horseradish peroxidase Immunization Irdye 800 Mitochondrial Mouse Novus Peptide Rabbit Rabbits Synuclein Tomm20

Corresponding Organization :

Other organizations : Eli Lilly (United States), University of Helsinki, International Institute of Molecular and Cell Biology, University of Pittsburgh, Institute for Neurodegenerative Disorders, Great Ormond Street Hospital, University College London, University of Cambridge, University of Sheffield, Eli Lilly (United Kingdom)

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Variable analysis

independent variables

Mitochondrial primary antibodies: TOMM20 (Santa Cruz), TIMM9 (Abcam), NDUFA9 (Abcam), COX4i1 (Abcam), ATP5A (Abcam)
LC3 primary antibodies: Rabbit anti-LC3 (Novus Biologicals; NB100–2220) used at 1:1000 dilution
Beta- and Gamma-synuclein primary antibodies: mAb α/β-Synuclein (Syn205, Cell Signaling; 1:1000) or pAb γ1-Synuclein (1:1000)

dependent variables

Protein expression levels or localization as detected by western blotting

control variables

β-ACTIN (Sigma-Aldrich) as a loading control
Mouse anti-actin (Sigma A5316) used at 1:500 dilution as a loading control
Polyclonal goat anti-rabbit immunoglobulins/HRP (Dako P0448) used at 1:5000 as a secondary antibody
Polyclonal goat anti-mouse immunoglobulins/HRP (P0447) used at 1:5000 as a secondary antibody
IRDye-800 and IRDye-680 (LI-COR, Lincoln, NE) conjugated secondary antibodies (1:10 000) for Odyssey Infrared Imaging

positive controls

None specified

negative controls

None specified

Annotations

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