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5 protocols using anti lipoic acid

1

Immunohistochemistry Reagents and Antibodies

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Monoclonal Anti-actin antibody, anti-fatty acid synthase (FAS), Prussian Blue, sodium pantothenate and trypsin were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-mitochondrial acyl carrier protein (mtACP), anti-aminoadipate semialdehyde dehydrogenase phosphopantetheinyl transferase (AASDHPPT), anti-NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 (NDUFA9), anti-NADH-ubiquinone oxidoreductase chain 1 (MTND1), DAPI and Hoechst 3342, were purchased from Invitrogen/Molecular Probes (Eugene, OR). NFS1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PANK2, complex 1 activity kit, PDH activity kit and aconitase activity kit were purchased from Abcam (Cambridge, UK). Anti-mitochondrial 10-formyltetrahydrofolate dehydrogenase (ALDH1L2), anti-cytosolic 10-formyltetrahydrofolate dehydrogenase (ALDH1L1), anti-alpha-aminoadipic semialdehyde synthase, mitochondrial (AASS), anti-LYRM4, anti-Tau clone HT7, anti-ISD11, anti-PANK1 and anti-PANK3 were purchased from Thermo-Fisher (Waltham, MA). Anti-lipoic acid was acquired from Merck (Darmstadt, Germany). A cocktail of protease inhibitors (complete cocktail) was purchased from Boehringer Mannheim (Indianapolis, IN). The Immun Star HRP substrate kit was from Bio-Rad Laboratories Inc. (Hercules, CA).
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2

Monoclonal Antibody-Based Mitochondrial Profiling

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Monoclonal Anti-actin antibody, anti-fatty acid synthase (FAS), Prussian Blue, sodium pantothenate and trypsin were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-mitochondrial acyl carrier protein (mtACP), anti-aminoadipate semialdehyde dehydrogenase phosphopantetheinyl transferase (AASDHPPT), anti-NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 (NDUFA9), anti-NADH-ubiquinone oxidoreductase chain 1 (MTND1), DAPI and Hoechst 3342, were purchased from Invitrogen/Molecular Probes (Eugene, OR). NFS1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PANK2, complex 1 activity kit, PDH activity kit and aconitase activity kit were purchased from Abcam (Cambridge, UK). Antimitochondrial 10-formyltetrahydrofolate dehydrogenase (ALDH1L2), anti-cytosolic 10-formyltetrahydrofolate dehydrogenase (ALDH1L1), anti-alpha-aminoadipic semialdehyde synthase, mitochondrial (AASS), anti-LYRM4, anti-Tau clone HT7, anti-ISD11, anti-PANK1 and anti-PANK3 were purchased from Thermo-Fisher (Waltham, MA). Anti-lipoic acid was acquired from Merck (Darmstadt, Germany). A cocktail of protease inhibitors (complete cocktail) was purchased from Boehringer Mannheim (Indianapolis, IN). The Immun Star HRP substrate kit was from Bio-Rad Laboratories Inc. (Hercules, CA).
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3

Mitochondrial Dysfunction Biomarker Assay

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Sudan Black, Prussian Blue, ( ±) α-LA, Luperox® DI (tert-Butyl peroxide), anti-fatty acid synthase (FAS), and trypsin were purchased from Sigma Chemical Co. (St. Louis, MO). BODIPY® 598/591 C11, MitoTracker Deep Red FM, DAPI, were purchased from Invitrogen/Molecular Probes (Eugene, OR). MitoPeDPP® was purchased from Dojindo Molecular Technologies, Inc. (Rockville,MD) Anti-PANK2, anti-MTND1, anti-NDUFA9, anti-NFS1, anti-ISCU, anti-LYRM4anti-NRF2, PDH hand complex I activity kit and aconitase kit were purchased from Abcam (Cambridge, UK), Anti-mitochondrial 10-formyltetrahydrofolate dehydrogenase (ALDH1L2), anti-alpha-aminoadipic semialdehyde synthase (AASS), anti-FOXN4, anti-hnRNPA/B,anti-NF-Y, anti-Tau, anti-GPX4 and anti-AASDHPPT were purchased from Thermo-Fisher (Waltham, MA). Anti-lipoic acid was acquired from Merck (Darmstadt, Germany). Anti-PLA2G6 and anti-SOD were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-actin was acquired from MyBiosource (San Diego, California, USA). OxyBlot Protein Oxidation Detection Kit was acquired from Merck (Darmstadt, Germany). A cocktail of protease inhibitors (complete cocktail) was purchased from Boehringer Mannheim (Indianapolis, IN). The Immun Star HRP substrate kit was from Bio-Rad Laboratories Inc. (Hercules, CA).
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4

Antibody Sources for Mitochondrial Proteins

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IBA57, NFU1, and CIAO3 were detected by polyclonal antisera raised in rabbits against recombinant human full-length proteins purified from E. coli (Navarro-Sastre et al., 2011 (link); Sheftel et al., 2012 (link); Stehling et al., 2018a (link)). Purified polyclonal rabbit antibodies directed against UQCRFS1, UQCRC2, COX2, COX6A/B, and F1α/β were a kind gift from H. Schägger and I. Wittig (Frankfurt, Germany). Purified rabbit anti-GPAT was kindly provided by H. Puccio (Illkirch, France), and mouse anti-IRP1 was a kind donation from R. Eisenstein (Madison, United States). Mouse monoclonal antibodies directed against NDUFS1, NDUFS8, NDUFV2, NTHL1, or β-actin, as well as rabbit anti-DPYD, were availed from Santa Cruz Biotechnology. Mouse monoclonal antibodies directed against NDUFA9, NDUFA13, NDUFB4, NDUFB6, or SDHB were obtained from MitoSciences via Abcam. Mouse anti-DLAT (Cell Signaling Technology, 4A4-B6-C10), anti-GAPDH (Calbiochem), and anti–α-tubulin (clone DM1a, Sigma Aldrich) and rabbit anti-ACO1 (Invitrogen), anti-lipoic acid (Merck/Calbiochem), or anti-POLD1 (PTG Lab) were also commercially available.
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5

Western Blot Analysis Protocol

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For Western blot analysis, cells were dissolved in 2× Laemli buffer, sonificated, and boiled for 5 min with 5% β‐mercaptoethanol (Sigma). Protein concentration was determined using DC protein assay (Bio‐Rad). Equal amounts of protein (10–30 μg) were loaded on a 10, 12, or 4–20% gradient gel (Bio‐Rad), and transferred onto PVDF membranes using the Trans Blot Turbo System (Bio‐Rad). Membranes were blocked in 5% fat‐free milk for 1 h at room temperature, and then rinsed in PBS‐Tween 20. Incubations with primary antibodies were done overnight at 4°C followed by incubations with HRP‐conjugated secondary antibodies (Amersham 1:5,000) for 1.5 h at room temperature. Detection was performed using ECL reagent (Thermoscientific) and visualized using the ChemiDoc imager (Bio‐Rad). The following primary antibodies were used: anti‐mtACP antibody (Abcam, 1:1,000), anti‐lipoic acid (Merck, 1:1,000), anti‐PDH‐E2 (Abcam, 1:1,000), anti‐α‐Tubulin (Sigma, 1:5,000), anti‐GAPDH (Fitzgerald, 1:10,000), anti‐dPank/Fbl (Bosveld et al, 2008; 1:1,000), and anti‐hPANK2 (Origene 1:500). A complete list of antibodies used can be found in Appendix Table S3.
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