Western Blot Analysis of Mitochondrial Proteins

Commercially available antibodies were used in western blots (WBs), for the detection of OPA1 and TIM44 (BD Transduction Laboratories, Franklin Lakes, New Jersey, USA), OMA1 (Novus Biologicals, Littleton, Colorado, USA), tubulin (DSHB, University of Colorado, Colorado, USA), HSP60 (Enzo Life Science, Farmingdale, New York, USA), MTCO1 and VDAC1 (Abcam, Cambridge, UK). Anti-AFG3L2 N-terminal (OriGene, Rockville, Maryland, USA) and an anti-AFG3L2 C-terminal antibody (previously generated in the lab)7 (link) were used to detect AFG3L2. Secondary antibodies used included Horseradish Peroxidase (HRP)-conjugated antimouse and antirabbit IgG (Amersham Bioscience, Buckinghamshire, UK). Chloramphenicol succinate sodium salt (Merck, KGaA, Billerica, Massachusetts, USA) was used at a concentration of 200 µg/mL for 24 hours. Carbonyl cyanide-4 -(trifluoromethoxy)phenylhydrazone (FCCP) (Merck) was used at a concentration of 10 µM for 10 min.

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Tulli S., Del Bondio A., Baderna V., Mazza D., Codazzi F., Pierson T.M., Ambrosi A., Nolte D., Goizet C., Toro C., Baets J., Deconinck T., DeJonghe P., Mandich P., Casari G, & Maltecca F. (2019). Pathogenic variants in the AFG3L2 proteolytic domain cause SCA28 through haploinsufficiency and proteostatic stress-driven OMA1 activation. Journal of Medical Genetics, 56(8), 499-511.

Publication 2019

TIM44 HSP60 MTCO1 VDAC1

Anti c antibody Antibodies Biologicals Carbonyl cyanide Chloramphenicol succinate sodium Fccp Horseradish peroxidase Novus Opa1 Phenylhydrazone Salt Tubulin Vdac1 Western blots

Corresponding Organization : Vita-Salute San Raffaele University

Other organizations : Cedars-Sinai Medical Center, University of Giessen, Inserm, Office of the Director, National Institutes of Health, Antwerp University Hospital, University of Antwerp, University of Genoa, Ospedale Policlinico San Martino, Telethon Institute Of Genetics And Medicine

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Variable analysis

independent variables

Chloramphenicol succinate sodium salt (200 µg/mL for 24 hours)
Carbonyl cyanide-4 -(trifluoromethoxy)phenylhydrazone (FCCP) (10 µM for 10 min)

dependent variables

Detection of OPA1, TIM44, OMA1, tubulin, HSP60, MTCO1, VDAC1, and AFG3L2 (N-terminal and C-terminal) by Western blot

control variables

Commercially available antibodies used for Western blot detection
Horseradish Peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgG secondary antibodies

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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