The polyclonal antibody against human Lon synthetic peptide (CRRPQDKDAKGDKDG) was raised in rabbit and affinity purified by GenScript (Nanjing, China). The mouse monoclonal antibody against TFAM was purchased from Abcam (ab176558). The goat polyclonal β-actin antibody was from Santa Cruz (sc-1616). TMP was purchased from Sigma (W323713). Biotinylated TMP was synthesized in our laboratory (see Supplementary data). CDDO was kindly provided by Dr Michael Sporn (Dartmouth University, NH). Bortezomib (Vaecade) was from LC Laboratories (B-1408). All other chemicals used were of highest analytical grade and obtained from Sigma, unless otherwise stated.
Ab176558
Manufactured by Abcam
Sourced in United Kingdom
Ab176558 is a laboratory equipment product from Abcam. It serves as a core function, providing a specific capability for use in various research and experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880, by Zeiss (4 mentions)
Mitotrackertm deep red fm, by Thermo Fisher Scientific (2 mentions)
Ab154856, by Abcam (1 mentions)
Ab11175, by Abcam (1 mentions)
Ab46798, by Abcam (1 mentions)
Ab6556, by Abcam (1 mentions)
Anti myc 9e10, by Merck Group (1 mentions)
Ab125001, by Abcam (1 mentions)
Ab55744, by Abcam (1 mentions)
Ab176328, by Abcam (1 mentions)
Ab182858, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab175932, by Abcam (1 mentions)
Ab191838, by Abcam (1 mentions)
Ab65080, by Abcam (1 mentions)
Ab269513, by Abcam (1 mentions)
Ab205531, by Abcam (1 mentions)
Ab125066, by Abcam (1 mentions)
Donkey anti rabbit igg h l, by Abcam (1 mentions)
F1804, by Merck Group (1 mentions)
7 protocols using ab176558
1
Polyclonal Antibody Production and Characterization
2
Antibody Generation and Validation Protocol
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A polyclonal antibody against ASPH was raised in our institution using the synthetic peptide antigen of 12 amino-acid residues around the Fe2+-binding domain of ASPH. The antibodies against mitochondrial biomarkers heat shock protein 60 (ab46798) and voltage-dependent anion channel (ab154856), endoplasmic reticulum biomarkers calnexin (ab195198), green fluorescent protein (ab6556), H2AX (ab11175), POLG (ab207558) and mtTFA (ab176558) were purchased from Abcam (Cambridge, UK). The mouse monoclonal anti-myc (9E10) and HA antibodies (12CA5) were purchased from Sigma-Aldrich (St Louis, MO, USA) and Roche (Basel, Switzerland), respectively. Additional information on secondary antibodies is detailed in the Supplementary Information .
3
Western Blot Analysis of Cardiac Proteins
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The left ventricular protein was extracted with RIPA lysate containing protease inhibitors, and the protein concentration was determined by BCA method. The SDS-PAGE was performed, electrophoresed to nitrocellulost membrane, and blocked with 5% bovine serum albumin for 1 h. Next, antibodies and dilutions as follows: Bax (Abcam, ab32503, rabbit), Bcl-2 (Abcam, ab182858, rabbit), PGC-1α (Abcam, ab176328, rabbit), PPAR-α (Abcam, ab245119, rabbit), RXR-α (Abcam, ab125001, rabbit), NRF-1 (Abcam, ab55744, rabbit) and Tfam (Abcam, ab176558, rabbit).The fluorescently labeled secondary antibodies are then conjugated by incubation. The blotted proteins were examined and quantified on the Odyssey Infrared Imaging system. The ratio of the target protein to the internal reference protein β-actin reflects the relative expression of the protein.
4
Mitochondrial Dynamics Visualization
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RSC96 cells (1^105) in a 30 mm confocal dish were incubated with MitoTracker TM Deep Red FM (200 nM, M22426, Thermo Fisher) for 30 min at 37°C in the dark. Then, the cells were fixed by 4% paraformaldehyde, permeabled membrane by 0.1% Triton X-100, and blocked by 3% BSA. The cells were incubated overnight with mtTFA antibody (1 : 500, ab176558, Abcam). In the next day, the cells incubated with the secondary antibody donkey anti-rabbit IgG-H&L (Alexa Fluor 488). DAPI was used to stain cell nuclei. Fluorescent images were captured by Zeiss LSM 880 with Airyscan. Images were analyzed using Image-Pro Plus 6.0 software.
5
Mitochondrial Metabolic Modulation
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Anti-peroxisome proliferator-activated receptor γ coactivator 1 alpha (PCG-1α) antibody (ab191838), anti-nuclear respiratory factor 1 (NRF1) antibody (ab175932), anti-mitochondrial transcription factor A (TFAM) antibody (ab176558), anti-ferritin heavy chain 1 (FTH1) antibody (ab65080), anti-transferrin receptor (ab269513), anti-glutathione peroxidase 4 antibody (ab125066) and anti-Grsf1 (ab205531) were purchased from Abcam. Rifampicin (R817237), isoniazid (I811711), and silybin (S817883) were purchased from Macklin Inc.
6
Mitochondrial DNA Detection via TFAM
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mtDNA was detected by expression of the mtDNA-binding protein TFAM (transcription factor A, mitochondrial) (Mcarthur et al., 2018 (link)). Cells plated in a 20-mm confocal dish and incubated with MitoTracker TM Deep Red FM (200 nM, M22426, Thermo Fisher), followed by fixing with 4% paraformaldehyde for 10 min. 0.1% Triton X-100 for 10 min and 3% bovine serum albumin (BSA)/PBS for 30 min were used to permeabilize and block the nonspecific binding sites. Then, cells were incubated with the mtTFA antibody (1:500, ab176558, Abcam) and donkey anti-rabbit IgG-H&L (Alexa Fluor 488) in 1.5% BSA. Fluorescent images were captured by a Zeiss LSM 880 with Airyscan. Images were analyzed using Image-Pro Plus 6.0 software.
7
Comprehensive Antibody Panel for Protein Analysis
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For immunofluorescence, western blot analysis or ChIP-qPCR, the following antibodies were used in these experiments: TFAM (ab89818 or ab176558, Abcam), amphiregulin (AF262, R&D Systems), beta tubulin (66240–1-Ig, Proteintech), c-JUN (H-79) (sc-1694, Santa Cruz Biotechnology) or c-JUN (G-4) (sc-74543), phospho-c-Jun (Ser73) (D47G9) XP rabbit mAb (#3270, Cell Signaling Technology), YAP/TAZ (D24E4) (#8418) or YAP (D8H1X) XP® rabbit mAb (#14074), phospho-YAP (Ser127) (D9W2I) rabbit mAb (#13008), purified mouse anti-TEF-1 (610923, BD Biosciences), TFIID (TBP) (58C9) (sc-421), BRG-1 (G-7) (sc-17796), anti-FLAG M2 monoclonal, affinity purified (F1804, Sigma-Aldrich), anti-histone H3 (mono methyl K4) - ChIP grade (ab8895) or anti-histone H3 (acetyl K27) - ChIP grade (ab4729).
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