Ab13534, by Abcam - Product details

1

Acetylation of Mitochondrial SOD2 Quantification

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Protein samples (10–60 µg) from ethanol-fed and pair-fed controls were separated using 12% SDS-PAGE at 150 V for 1 hour, then transferred to activated Hybond-PVDF membrane (GE Healthcare, Buckinghamshire, UK). Membranes were blocked using 5% (w/v) non-fat dry milk in Tris-Buffered Saline-Tween 20 TBS/0.1 % (v/v) Tween (TBS-T) for 1 hour at room temperature. Membranes were incubated with primary antibodies against SOD2 (ab13534), SOD2 acetyl K68 (ab137037), or SOD2 acetyl K122 (ab214675) (Abcam, Cambridge, MA) overnight at 4°C, then washed three times with TBS-T, and incubated with horseradish peroxidase conjugated secondary antibody at room temperature for one hour. Membranes were washed again with TBS-T three times and then Clarity Western ECL Substrate (BioRad) was applied before imaging via Chemidoc® MP (Bio-Rad, Hercules, CA). 2,2,2-trichloroethanol (Sigma, Saint Louis, MO) stain was used to visualize overall protein load.
Two-dimensional SDS-PAGE was performed using 200 µg of liver whole cell extract and was separated on IPG strips (pH 3–11) (Bio-Rad), then resolved using 10% SDS-PAGE. The proteins were then transferred onto PVDF membrane and probed with anti-SOD2 (ab13534) and SOD2 acetyl-K68 (ab137037) antibody (Abcam, Cambridge, MA) using standard Western blot analysis as described above.

Assiri M.A., Roy S.R., Harris P.S., Ali H., Liang Y., Shearn C.T., Orlicky D.J., Roede J.R., Hirschey M.D., Backos D.S, & Fritz K.S. (2017). Chronic Ethanol Metabolism Inhibits Hepatic Mitochondrial Superoxide Dismutase via Lysine Acetylation. Alcoholism, clinical and experimental research, 41(10), 1705-1714.

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2

Immunohistochemical Analysis of Liver Tissue

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Freshly excised liver tissues were fixed in 10% formalin (Sigma, Saint Louis, MO) overnight. The tissue was then embedded in paraffin, cut and mounted on slides by the University of Colorado Anschutz Medical Campus Histology Core. Standard hematoxylin and eosin (H&E) staining was performed. Immunohistochemistry was performed using the ABC citrate antigen retrieval system (Vector labs, Burlingame, CA) and by incubating liver tissue sections with primary antibodies raised against CYP2E1 (AB1252; Millipore, Darmstadt, Germany), SOD2 (ab13534; Abcam, Cambridge, MA, USA), and SOD2 acetyl-K68 (ab137037; Abcam, Cambridge, MA, USA) overnight at 4 °C via a protocol described by Harris et. al. (Harris et al., 2015 (link)). Histologic images were captured on an Olympus BX51 microscope equipped with a 4 megapixel Macrofire digital camera using the PictureFrame Application 2.3 (Optronics). All images were cropped and assembled using Photoshop CS2 (Adobe Systems, Inc.). Images were then imported into Slidebook (3I, Denver CO) for quantification, which was performed on 5 images per animal, 3–4 animals per group.

Assiri M.A., Roy S.R., Harris P.S., Ali H., Liang Y., Shearn C.T., Orlicky D.J., Roede J.R., Hirschey M.D., Backos D.S, & Fritz K.S. (2017). Chronic Ethanol Metabolism Inhibits Hepatic Mitochondrial Superoxide Dismutase via Lysine Acetylation. Alcoholism, clinical and experimental research, 41(10), 1705-1714.

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3

Protein Isolation and Western Blotting

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Cells were homogenized in RIPA buffer supplemented with protease and phosphatase inhibitors and centrifuged at 15,000 g for 15 min at 4 °C. Protein concentration was determined using the BCA protein assay kit (Thermo Scientific, Rockford, IL, USA), and equal amounts of protein (20–30 μg) were subjected to 8–12% SDS-PAGE and transferred to an Immobilon membrane (Millipore, Bedford, CA, USA). Blots were treated with primary antibodies, followed by the corresponding secondary antibody with horseradish peroxidase activity. Blots were developed using Pierce ECL reagent (Thermo Fisher Scientific, Waltham, MA, USA) in a Fujifilm LAS 3000 Intelligent Dark Box IV imaging system (Tokyo, Japan).
The following primary antibodies were used: anti-PEPCK-M (ab70359, Abcam, Cambridge, UK), anti-PEPCK-C (generous gift of Dr. Daryl K. Granner, Vanderbilt University, Nashville, TN, USA), anti-SOD2 (ab13534, Abcam), anti-p53 (ab26, Abcam), anti-p21 (sc397, Santa Cruz Biotechnology, Dallas, TX, USA), anti-gamma tubulin (T6557, Sigma-Aldrich, St. Louis, MO, USA), and anti-PKC-ζ (9372S, Cell Signaling Technology, Danver, MA, USA).

Hyroššová P., Aragó M., Moreno-Felici J., Fu X., Mendez-Lucas A., García-Rovés P.M., Burgess S., Figueras A., Viñals F, & Perales J.C. (2021). PEPCK-M recoups tumor cell anabolic potential in a PKC-ζ-dependent manner. Cancer & Metabolism, 9, 1.

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4

Protein Expression Analysis in Clinical Samples

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The RIPA lysis buffer (Beyotime, China) was used extract total proteins from clinical tissues and cell lines. The proteins were separated by 10% sodium sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the targeted protein bands were transferred onto PVDF membrane (Sigma, USA). Subsequently, the PVDF membranes were probed with the primary antibodies including anti-β-actin (#ab8226, Abcam, USA), anti-SOD2 (#ab13534, Abcam, USA), anti-NLRP3 (#ab214185, Abcam, USA), anti-CyclinD1 (#ab16663, Abcam, USA), anti-Cyclin E2 (#ab32103, Abcam, USA), anti-CDK2 (#ab32147, Abcam, USA), anti-CDK4 (#ab108357, Abcam, USA), anti-CDK6 (#ab124821, Abcam, USA), anti-Caspase 3 (#ab13847, Abcam, USA), anti-Bax (#ab32503, Abcam, USA), anti-Bcl-2 (#ab185002, Abcam, USA), anti-Caspase 1 (#ab238979, Abcam, USA) and anti-IL-1β (#ab33774, Abcam, USA). After that, the membranes were incubated with anti-Rabbit IgG antibody at 4°C for 24h, and the enhanced chemiluminescent (ECL) system was employed to detect the protein bands. Finally, all the protein bands were quantified by Image J software. The primers sequences of the target genes were listed in Table 1.

Liu J., Yao L., Zhang M., Jiang J., Yang M, & Wang Y. (2019). Downregulation of LncRNA-XIST inhibited development of non-small cell lung cancer by activating miR-335/SOD2/ROS signal pathway mediated pyroptotic cell death. Aging (Albany NY), 11(18), 7830-7846.

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5

Quantitative Mitochondrial Protein Analysis

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For each specimen, four thoraxes were dissected and homogenized by dounce tissue grinder in RIPA buffer containing protease inhibitor (cOmplete^TM, Roche). Cellular debris were removed by centrifugation at 4 °C, 14000× g for 20 min. The supernatants were collected and the protein concentrations were determined by Pierce protein assay (Pierce 660 nm Protein Assay Reagent, ThermoScientific). 0.6 μg/well of proteins were loaded for SDS-PAGE and western-blot analysis.
The primary antibodies and the dilutions were as follows: mouse anti-ATP5A (50000x, abcam ab14748), mouse anti-Cytochrome C (10000x, abcam ab13575), mouse anti-PDHA1 (1000x, abcam ab110334), rabbit anti-SOD2 (10000x, abcam ab13534) and rabbit anti-alpha Tubulin (10000x, abcam ab18251). The secondary antibodies and the dilutions were as follows: anti-mouse IgG-HRP (2000x, Invitrogen 62–6520), or anti-rabbit IgG-HRP (5000x, abcam ab97051). The signals were developed by chemiluminescent HRP substrate (Immobilon^TM Western, Millipore) and recorded by a digital multispectral imaging system (BioSpectrum, UVP).

Jiang Y.F., Lin S.S., Chen J.M., Tsai H.Z., Hsieh T.S, & Fu C.Y. (2017). Electron tomographic analysis reveals ultrastructural features of mitochondrial cristae architecture which reflect energetic state and aging. Scientific Reports, 7, 45474.

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6

Immunofluorescent Staining of SOD2 in Cells

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Cells grown on glass coverslips precoated with bovine collagen (1μg/ml; Organogenesis, Canton MA) were fixed in 3% formaldehyde for 20 min, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 5% bovine serum albumin for 1 hr. Cells were incubated with anti-SOD2 (1:100; ab13534, Abcam, Cambridge, UK) overnight at 4°C, and then with Rabbit-Cy2-conjugated secondary antibody (1: 600; Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. Nuclei were counterstained by DAPI (1:10,000; Invitrogen). Stained objects were imaged with a Leica TCS SP8 confocal microscope using LAS software (Leica Microsystems, Buffalo Grove, IL).

Kinugasa H., Whelan K.A., Tanaka K., Natsuizaka M., Long A., Guo A., Chang S., Kagawa S., Srinivasan S., Guha M., Yamamoto K., St. Clair D.K., Avadhani N.G., Diehl J.A, & Nakagawa H. (2015). Mitochondrial SOD2 regulates epithelial-mesenchymal transition and cell populations defined by differential CD44 expression. Oncogene, 34(41), 5229-5239.

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7

Immunofluorescent Staining of SOD2 in Cells

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Cells grown on glass coverslips precoated with bovine collagen (1μg/ml; Organogenesis, Canton MA) were fixed in 3% formaldehyde for 20 min, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 5% bovine serum albumin for 1 hr. Cells were incubated with anti-SOD2 (1:100; ab13534, Abcam, Cambridge, UK) overnight at 4°C, and then with Rabbit-Cy2-conjugated secondary antibody (1: 600; Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. Nuclei were counterstained by DAPI (1:10,000; Invitrogen). Stained objects were imaged with a Leica TCS SP8 confocal microscope using LAS software (Leica Microsystems, Buffalo Grove, IL).

Kinugasa H., Whelan K.A., Tanaka K., Natsuizaka M., Long A., Guo A., Chang S., Kagawa S., Srinivasan S., Guha M., Yamamoto K., St. Clair D.K., Avadhani N.G., Diehl J.A, & Nakagawa H. (2015). Mitochondrial SOD2 regulates epithelial-mesenchymal transition and cell populations defined by differential CD44 expression. Oncogene, 34(41), 5229-5239.

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8

Immunohistochemical Analysis of Oxidative Stress Markers

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After dewaxing and hydration, the sections were placed in citrate buffer and heated to 95°C for 20 min. After the sections cooled naturally, we treated the sections with 3% H₂O₂ for half an hour. After washing the sections with phosphate-buffered saline (PBS), we treated the sections with 10% goat serum for 1 h. We then treated the sections with SOD1 (ab13498, Abcam, Cambridge, MA, USA), SOD2 (ab13534, Abcam, Cambridge, MA, USA), NF-E2-realted factor2 (Nrf2) (ab137550, Abcam, Cambridge, MA, USA), caspase8 (ab25901, Abcam, Cambridge, MA, USA), and caspase9 (ab32539, Abcam, Cambridge, MA, USA) antibodies at 4°C overnight. The next day, we treated the sections with the universal secondary antibody from the IHC staining kit (GeneTech, Shanghai, China) for 1 h. Finally, we used the developer in the IHC staining kit for color development.

Li H., Yang H., Wang D., Zhang L, & Ma T. (2020). Peroxiredoxin2 (Prdx2) Reduces Oxidative Stress and Apoptosis of Myocardial Cells Induced by Acute Myocardial Infarction by Inhibiting the TLR4/Nuclear Factor kappa B (NF-κB) Signaling Pathway. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 26, e926281-1-e926281-11.

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9

Immunohistochemical Analysis of Oxidative Stress

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The prepared paraffin slices were roasted, dewaxed and citrated buffer high pressure repaired, then they were naturally cooled, removed endogenous peroxidase by adding 3% hydrogen peroxide, and incubated with different primary antibody (SOD1, ab13498, 1 : 200, Abcam, USA; SOD2, ab13534, 1 : 200, Abcam, USA; CHOP, #2895, 1 : 200, CST, USA; caspase-3, ab32351, Abcam, USA; 1 : 200) in a wet box at 4°C overnight. The negative control group was replaced with PBS instead of the primary antibody. Washing in PBS three times for 5 minutes each time, secondary antibody (ALexar Fluor647, ab150079, 1 : 300, Abcam, USA) was incubated in a cassette for 1 h at 37°C, and then washed with PBS again. At last, Nuclear was counterstained with DAPI for 2 min and rinsed 3 times by PBS. The appropriate amount of antifluorescence-attenuating sealer (Solarbio, Shanghai, China) was used for sealing. Five different fields of fluorescence were observed under a conjugated double-focus microscope (Nikon, Japan). The fluorescence density was analyzed by Image-Pro Plus 6.0 software.

Hu W., Wang H., Shu Q., Chen M, & Xie L. (2020). Green Tea Polyphenols Modulated Cerebral SOD Expression and Endoplasmic Reticulum Stress in Cardiac Arrest/Cardiopulmonary Resuscitation Rats. BioMed Research International, 2020, 5080832.

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10

Protein Expression Analysis by Western Blot

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The cell homogenates were loaded into 10% SDS-polyacrylamide gels and subjected to electrophoresis. The separated proteins were transferred to nitrocellulose membranes and then incubated with bovine albumine serum solution for 1 hour, followed by overnight incubation at 4°C with the following antibodies: mouse monoclonal β-actin antibody (diluted 1:5000; AC5441 –Sigma Aldrich, St. Louis, MO, USA), goat polyclonal CAT antibody (CAT; diluted 1:1000; sc34285—Santa Cruz Biotechnology Inc., Dallas, TX, USA), rabbit polyclonal PGC-1α (dilution 1:2500; ab191838—Abcam, Cambridge, UK); rabbit polyclonal SOD2 (diluted 1:2000; ab13534—Abcam, Cambridge, UK), or with rabbit polyclonal SIRT3 (diluted 1:1500; ab189860—Abcam, Cambridge, UK). Protein detection was carried out using secondary infrared fluorescent dye conjugated antibodies absorbing at 800 nm or 700 nm. The blots were visualized using an Odyssey Infrared Imaging Scanner (Li-Cor Inc., Lincoln, USA).

Rezzani R., Favero G., Ferroni M., Lonati C, & Moghadasian M.H. (2019). A carnosine analog with therapeutic potentials in the treatment of disorders related to oxidative stress. PLoS ONE, 14(4), e0215170.

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Ab13534

Lab products found in correlation

16 protocols using ab13534

Acetylation of Mitochondrial SOD2 Quantification

Immunohistochemical Analysis of Liver Tissue

Protein Isolation and Western Blotting

Protein Expression Analysis in Clinical Samples

Quantitative Mitochondrial Protein Analysis

Immunofluorescent Staining of SOD2 in Cells

Immunofluorescent Staining of SOD2 in Cells

Immunohistochemical Analysis of Oxidative Stress Markers

Immunohistochemical Analysis of Oxidative Stress

Protein Expression Analysis by Western Blot

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