Protein Isolation and Western Blotting

Protein isolation and Western blotting were conducted essentially as described previously^{10 (link)}. 10 μg protein aliquots were electrophoresed on 4–20% TGX stain-free gels (Bio-Rad) and transferred to PVDF membranes using Trans-Blot® Turbo^TM PVDF Transfer pack (Bio-Rad). Primary antibodies and their dilutions were as specified previously^{10 (link)}, plus the following: porin (Abcam ab15895, rabbit polyclonal, 1:1,000), OXPHOS cocktail (Abcam ab110413, formerly Mitosciences ms604, mouse monoclonals for single subunits of each OXPHOS complex, namely NDUFB8, SDHB, UQCRC2, MTCOI and ATP5A, 1:250), AOX (21^st Century Biochemicals, customized rabbit polyclonal, 1:40,000), HSP60 (Abcam ab46798, rabbit polyclonal, 1:20,000), GAPDH (Cell Signaling #2118, rabbit polyclonal, 1:1,000) and LC3b (Novus NB600-1384, rabbit polyclonal, 1:1,000). Secondary antibodies and their dilutions were: goat anti-mouse IgG (Jackson ImmunoResearch #115-035-146, 1:10,000) and goat anti-rabbit IgG (Jackson ImmunoResearch #111-035-144, 1:20,000). Blot images have been cropped, rotated and framed where appropriate, and optimized for contrast and brightness, with reprobings of the same gel or different regions of the same gel shown as distinct image panels separated by white spacers. No other manipulations were introduced.

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Dhandapani P.K., Begines-Moreno I.M., Brea-Calvo G., Gärtner U., Graeber T.G., Javier Sanchez G., Morty R.E., Schönig K., Hoeve J.T., Wietelmann A., Braun T., Jacobs H.T, & Szibor M. (2019). Hyperoxia but not AOX expression mitigates pathological cardiac remodeling in a mouse model of inflammatory cardiomyopathy. Scientific Reports, 9, 12741.

Publication 2019

4–20% TGX stain-free gels porin OXPHOS cocktail HSP60 GAPDH goat anti-mouse IgG goat anti-rabbit IgG

Anti igg Antibodies Dilutions Gapdh Goat Isolation Membranes Mouse Novus Porin Protein Pvdf Rabbit Sdhb Stain Subunits

Corresponding Organization :

Other organizations : Tampere University, Universidad Pablo de Olavide, Centro Andaluz de Biología del Desarrollo, Instituto de Salud Carlos III, University of Giessen, Max Planck Institute for Heart and Lung Research, Central Institute of Mental Health, University of Helsinki

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein levels of porin, OXPHOS subunits, AOX, HSP60, GAPDH, and LC3b

control variables

Protein isolation and Western blotting methods described previously in reference 10
Primary and secondary antibody dilutions
Gel and membrane types used

controls

Positive controls: None explicitly mentioned
Negative controls: None explicitly mentioned

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