The total protein of lung tissues and cells was collected with RIPA buffer (Servicebio, Wuhan, China) with cocktail and phosphatase inhibitors (Thermo Fisher Scientific, Wuhan, China), followed by electrophoresed through 8–12% sodium dodecyl sulfate–polyacrylamide gel and then transferred to 0.45 μm PVDF membrane (Merck Millipore, USA). A total of 30 ug of protein was used for western blot experiments. After blocked with 5% milk, the membrane was immunoblotted with following primary antibodies(Except for labeled, all from Cell Signaling Technology, MA, USA) at 4 °C overnight: anti-ERRα (1:1000), anti-ZO-1 (1:1000; Abcam), anti-VE-cadherin (1:1000), anti-Occludin(1:1000), anti-JAM-A(1:1000; Abcam), anti-Sirt3 (1:1000), anti-Bcl-2 (1:1000), anti-Bax (1:1000), anti-Smac (1:1000), anti-Cytochrome c (1:1000), anti-caspase 3 (1:1000), anti-cleaved caspase 3 (1:500), anti-caspase 9 (1:1000), anti-cleaved caspase 9 (1:500), anti-LC3A/B(1:1000), anti-Beclin1 (1:1000), anti-p62 (1:1000), or anti-GAPDH (1:10000, Abcam, internal control). After incubated with the HRP-conjugated secondary antibodies at 25℃ for 1 h, the immunoblots were visualized using the ECL system. The gray value was analyzed using the Image J and calculate the relative protein level based on the density ratio of the target protein to GAPDH (internal control).
Anti sirt3
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Anti-Sirt3 is a laboratory product that can be used to detect and quantify the SIRT3 protein, which is a member of the sirtuin family of enzymes. SIRT3 is involved in the regulation of cellular metabolism and plays a role in various biological processes. This product can be utilized in research applications that require the identification and measurement of SIRT3 levels in different samples or experimental conditions.
Automatically generated - may contain errors
Lab products found in correlation
Anti occludin, by Abcam (2 mentions)
Anti bax, by Abcam (2 mentions)
Anti caspase 3, by Abcam (2 mentions)
Anti cleaved caspase 9, by Abcam (2 mentions)
Anti beclin1, by Abcam (2 mentions)
Anti caspase 9, by Abcam (2 mentions)
Anti cytochrome c, by Abcam (2 mentions)
Ripa buffer, by Wuhan Servicebio Technology (2 mentions)
Anti errα, by Abcam (2 mentions)
Anti bcl 2, by Abcam (2 mentions)
Anti gapdh, by Abcam (2 mentions)
0.45 μm pvdf membrane, by Merck Group (2 mentions)
Anti ve cadherin, by Abcam (2 mentions)
Anti zo 1, by Abcam (2 mentions)
Anti smac, by Abcam (2 mentions)
Anti lc3a b, by Abcam (2 mentions)
Except for labeled, by Cell Signaling Technology (2 mentions)
Anti jam a, by Abcam (2 mentions)
Anti p62, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
12 protocols using anti sirt3
1
Comprehensive Lung Protein Analysis
2
Protein Quantification and Western Blot Analysis
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Cells were scraped with PBS and lysed with ice-cold RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease inhibitor cocktail. The lysates were centrifuged at 12,000× g for 15 minutes at 4°C, and the precipitate was discarded. Equal amount protein (10 µg) was electrophoresed with sodium dodecyl sulfate-polyacrylamide gel and then the proteins were transferred to a polyvinylidene difluoride membrane (Merck Millipore, Kenilworth, NJ, USA). The primary antibodies used in this study included anti-Sirt3 (Abcam, #ab86671), anti-Ku70 (Invitrogen, #MA5-13110), anti-acetyl (Abcam, #ab80178), anti-BAX (Cell Signaling Technology, Danvers, MA, USA; #2774), anticytochrome c (Cell Signaling Technology, #4280), anticaspase 3 (Cell Signaling Technology, #9662), and anti-β-actin (Cell Signaling Technology, #3700). After incubation with primary and secondary antibodies, the immunoreactivity bands were visualized by using an enhanced chemiluminescence substrate kit (Thermo Scientific, Pittsburgh, PA, USA).22 (link)
3
SIRT1-7, TIP60, and GPAT3 Interaction Analysis
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The open reading frame of human SIRT1‐7, TIP60, and GPAT3 (Miaolingbio) were subcloned into pcDNA3.1‐3×flag or pcDNA3.1‐HA vectors. Transfected cells were lysed with NP‐40 lysis buffer (Biosharp, Anhui, China). Anti‐AcGPAT3 were custom‐made from GL (GL Biochem, Shanghai, China) and applied for IP and Co‐IP assays. The immune precipitants eluted from SureBeads™ Protein A&G Magnetic Beads (Bio‐Rad Inc., Hercules, CA, USA) were examined by immunoblotting as described.
32 (link) Anti‐rabbit and mouse IgG (Beyotime) were used as negative controls. Anti‐TIP60, anti‐SIRT3, anti‐GPAT3, anti‐Flag, and anti‐HA (1:1000) were obtained from Abcam (Cambridge, MA, USA).
32 (link) Anti‐rabbit and mouse IgG (Beyotime) were used as negative controls. Anti‐TIP60, anti‐SIRT3, anti‐GPAT3, anti‐Flag, and anti‐HA (1:1000) were obtained from Abcam (Cambridge, MA, USA).
4
Immunohistochemical Analysis of Liver Sirtuin Proteins
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Liver tissues were resected (2cm*2cm) and fixed in 10% formalin for 12 hours. Paraffin-embedded sections (4-μm thick) were prepared for immunohistochemical and hematoxylin and eosin (H&E) staining. Sections were incubated with the following primary antibody at 4°C overnight, anti-SIRT3 (Abcam, UK,1:300), anti-SIRT6 (Proteintech.,China,1:100), anti-SIRT7 (Abcam, UK,1:1000). After incubation with labeled the following day using an anti-rabbit universal two-step detection kit (Beijing Zhongshan Jinqiao Biotechnology Co. LTD, China). Finally, the signal was detected by DAB method (Beijing Zhongshan Jinqiao Biotechnology Co. LTD, China). Images were collected using NanoZoomer S60 C13210 series (S60 C13210, Hamamatsu Photonics K.K., Japan).
5
Subcellular Protein Extraction and Quantification
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Total protein was obtained using RIPA lysis buffer. Cell membrane, cytoplasm and mitochondrial proteins were obtained using the cell membrane protein and cytoplasmic protein extraction kit (Beyotime) and the cell mitochondrial isolation kit (Beyotime). BCA protein assay Kit (APPLYGEN) was used to quantitate the protein levels. The primary antibody information used is as follows: anti-CLDN10 (1:1000; Abcam, ab52234), anti-Flag (1:1000; CST, 14793S), anti-ATP5O (1:2000; Abcam, ab110276), anti-Acetyl-ATP5O (1:200; Abcam, ab214339), anti-SIRT3 (1:1000; Abcam, ab217319), anti-NDUFS2 (1:5000; Abcam, ab192022), anti-Cleaved-Caspase 3 (1:1000; Affinity, AF7022), anti-E-cadherin (1:10000; Abcam, ab40772), anti-N-cadherin (1:5000, Abcam, ab76011), anti-SDHB (1:5000, Proteintech, China), anti-GAPDH (1:8000, Proteintech, China), anti-ATP1A1 (1:8000, Proteintech, China) and anti-COX Ⅳ (1:8000, Proteintech, China).
6
Licorice-Derived Liquiritigenin Apoptosis
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Standard liquiritigenin of purity >98% was purchased from Aladdin Holdings Group Co., Ltd. (Shanghai, China). CP of over 98.5% purity (by HPLC) and the TUNEL kit were purchased from Beyotime Biotechnology (Shanghai, China). Annexin V-FITC/PI double staining apoptosis detection kit were obtained from BestBio (Shanghai, China). The antibodies used for Western blot analysis were as follows: anti-PGC-1α (Mouse, 1:1000, Catalog No.66369-1-Ig), anti-TFAM (Rabbit, 1:1000, Catalog No.22586-1-AP), anti-BCL-2 (Rabbit, 1:1000, Catalog No.26593-1-AP), anti-BAX (Mouse, 1:1000, Catalog No.60267-1-Ig), anti-β-actin (Mouse, 1:6000, Catalog No.66009-1-Ig), purchased from Protein Tech Group (Chicago, IL, USA), and anti-SIRT3 (Rabbit, 1:1000, Catalog No.ab189860), purchased from Abcam (Abcam, Cambridge, UK). Reagents related to cell culture such as culture medium, fetal bovine serum, and streptomycin and penicillin were purchased from Gibco (Shanghai, China).
7
Extracellular Vesicle Protein Analysis
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Protein lysates extracted from EVs, cells or mitochondria were electrophoretically separated using the 10% SDS-PAGE and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were then incubated with 5% BSA for 2 h at RT. Thereafter, they were exposed to primary antibodies listed in Additional file 1 : Table S1 overnight at 4 ℃. Following this, the membranes underwent incubation for 1 h with secondary HRP-conjugated antibodies at RT. The targeted signals were visualized using a ECL substrate (Pierce, #32209, USA).
For the CoIP assay, the treated HUVECs were collected and lysed in IP lysis buffer (Pierce, #87788, USA). Cell extracts were incubated with anti-sirt3 (Abcam, #ab246522, USA) and Pierce Classic Magnetic IP/Co-IP Beads (Pierce, #88804, USA). The immunoprecipitate was separated from the supernatant using a magnetic frame for magnetic beads and subsequently washed three times in PBS containing 0.05% NP-40. The proteins eluted from the beads were boiled in 1 lysed SDS gel-loading buffer and resolved by 10% SDS-PAGE. Primary antibody details were described in the Additional file1 : Table S1.
For the CoIP assay, the treated HUVECs were collected and lysed in IP lysis buffer (Pierce, #87788, USA). Cell extracts were incubated with anti-sirt3 (Abcam, #ab246522, USA) and Pierce Classic Magnetic IP/Co-IP Beads (Pierce, #88804, USA). The immunoprecipitate was separated from the supernatant using a magnetic frame for magnetic beads and subsequently washed three times in PBS containing 0.05% NP-40. The proteins eluted from the beads were boiled in 1 lysed SDS gel-loading buffer and resolved by 10% SDS-PAGE. Primary antibody details were described in the Additional file
8
Immunohistochemical Evaluation of Sirt3 in Glioma
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As described by others,19 (link) IHC was used to evaluate the Sirt3 protein expression in glioma tissues. Briefly, deparaffinized and rehydrated slides were treated in 10 mM citrate buffer (pH 6.0) at 95°C for antigen recovery, followed by quenching the endogenous peroxidase activity with 3% H2O2 incubation. Five percent FBS was used for blocking the nonspecific binding sites. Slides were then incubated with primary antibody (anti-Sirt3; Abcam, Cambridge, MA, USA; #ab86671; 1:200) at 4°C overnight. DAB staining kit (Tiangen, China) was used to detect the immunoreactivity according to the manufacturer’s instructions.
The level of Sirt3 was determined by the degree of staining intensity and the percentage of positively stained cells. Examination and scoring were performed by two pathologists independently. In brief, weak staining, moderate staining, and strong staining were scored as 1, 2, and 3, respectively. The percentages of positively stained cells were scored as follows: 1 for less than 20%, 2 for 20%–50%, and 3 for 50%–100%. The final IHC score was defined by multiplying the the two scores above, ranging from 1 to 9. Tissues with a final score no more than 4 were regarded as low-expression cases; otherwise, it will be grouped into the Sirt3 high-expression group.
The level of Sirt3 was determined by the degree of staining intensity and the percentage of positively stained cells. Examination and scoring were performed by two pathologists independently. In brief, weak staining, moderate staining, and strong staining were scored as 1, 2, and 3, respectively. The percentages of positively stained cells were scored as follows: 1 for less than 20%, 2 for 20%–50%, and 3 for 50%–100%. The final IHC score was defined by multiplying the the two scores above, ranging from 1 to 9. Tissues with a final score no more than 4 were regarded as low-expression cases; otherwise, it will be grouped into the Sirt3 high-expression group.
9
Mitochondrial Protein Interactions in Stress Response
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Cell lysates were resolved by SDS-PAGE, transferred onto PVDF membranes, and incubated with the following primary antibodies: anti-Urocortin3 (Ucn3) (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-Mfn2, anti-Idh2 (Abcam), anti-dehydrogenase E1 and transketolase domain containing 1 (Dhtkd1) (Abcam), anti-glutamate dehydrogenase 1 (Glud1) (Abcam), anti-Sirt3 (Abcam), and anti-GAPDH (Multisciences, Shanghai, China). The samples were incubated with horseradish peroxidase-conjugated secondary antibody. The blots were developed using enhanced chemiluminescence reagents (Affinity Biosciences, Cincinnati, OH, USA), and the density of the products was quantitated using the ImageJ software. For coimmunoprecipitation analysis, total protein extracts were immunoprecipitated with anti-Sirt3 antibody (ABclonal Technology, Hubei, China) and subjected to Western blot analysis by anti-Mfn2 antibody.
10
Western Blot Analysis of Sirt3 Protein
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Total protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with proteinase inhibitors. Protein concentration was determined by a BCA Protein Assay Kit (Beyotime, Shanghai, China). Then, 25 μg of protein sample was separated by 12% SDS-PAGE and transferred onto PVDF membranes (Burlington, MA, USA) by electroblotting, after which nonspecific binding to the membrane was blocked with 5% non-fat milk for 1–2 h at RT. Then, the blotted membranes were probed with anti-Sirt3 (1:2000, Abcam, Cambridge, MA, UK) and anti-β-actin (1:1500, Abcam, Cambridge, MA, UK) antibodies diluted in Tris-buffered saline (TBS) overnight at 4 °C. After incubating with antirabbit IgG secondary antibody (1:2000 dilution, Proteintech, Chicago, IL, USA), protein blots were visualized using an Electro-Chemi-Luminescence detection system (JENE, UK) and quantified with ImageJ software.
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