SIRT3 (TRCN 0000038892), HSPA9 (TRCN0000029450, TRCN0000029452), and scrambled shRNAs (in pLK0.1) were from Sigma (Cat # SHC002). p53 shRNA was described (Rodier et al., 2009 (link)). The AMPK-DN (D157A) (Zhou et al., 2009 (link)) cDNA was cloned into the DraI and XbaI sites of lentiviral vector 670-1 and packaged as described (Campeau et al., 2009 (link)).
Sirt3
Manufactured by Merck Group
Sourced in United States
SIRT3 is a lab equipment product manufactured by Merck Group. It is a protein that functions as an enzyme involved in cellular processes. The core function of SIRT3 is to regulate cellular metabolism and energy production within the mitochondria of the cell.
Automatically generated - may contain errors
Lab products found in correlation
Lysis buffer, by Biosharp (2 mentions)
Ecl western blotting reagent, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Sirt3, by Cell Signaling Technology (2 mentions)
Hsp60, by BD (2 mentions)
Mtco1, by Abcam (2 mentions)
Anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Foxo3a, by Cell Signaling Technology (2 mentions)
Radioimmunoprecipitation assay (ripa), by Thermo Fisher Scientific (1 mentions)
Lc3 1 2, by Merck Group (1 mentions)
Ampkα, by Cell Signaling Technology (1 mentions)
Ecl substrate, by Bio-Rad (1 mentions)
Sirt2, by Merck Group (1 mentions)
Pepck1, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated anti mouse and anti rabbit antibodies, by Cell Signaling Technology (1 mentions)
Sirt1, by Santa Cruz Biotechnology (1 mentions)
Mmp 9, by Abcam (1 mentions)
β catenin, by Abcam (1 mentions)
10 protocols using sirt3
1
Lentiviral Knockdown and Overexpression
2
Western Blot Analysis of HK-2 Cell Proteins
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After stimulation, HK-2 cells were washed twice with ice-cold PBS and lysed by RIPA (Thermo Fisher Scientific, Inc.) with phosphatase and protein inhibitor for 30 min on ice, followed by sonication twice. Then whole cell lysate was centrifuged at 4°C, and 16,000 × g for 10 min. The supernatants were collected for proteins analysis. Protein concentration in the supernatants was determined using the bicinchoninic acid (BCA) protein assay. Cell proteins (10 µg) were separated by 4-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membrane was blocked in 5% milk at room temperature for 2 h and then were incubated in primary antibodies against LC3-I/II (product no. ABC929; Sigma-Aldrich; Merck KGaA), SIRT3 (product no. 5490S), phosphorylated (p)-AMPKα (Thr172) (product no. 2535S), AMPKα (product no. 2603S), p62 (product no. 5114S) and β-actin (product no. 3700S) (all 1:1,000; from Cell Signaling Technology, Inc.) at 4°C overnight. Membranes were washed in TBS-T (0.01% Tween) 3 times and incubated with HRP-conjugated secondary antibody (goat anti-mouse; cat. no. BA1050 and goat anti-rabbit, cat. no. BA1054 both from BOSTER Biological Technology co. ltd.) at room temperature for 1 h. The signals were visualized with ECL substrate (Bio-Rad Laboratories, Inc.) and quantified using ImageJ 1.48V software (NIH).
3
Western Blot Analysis of Protein Expression
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Cultured cells were lysed with ice-cold lysis buffer (Biosharp). Extracted proteins were mixed with loading buffer containing 5% 2-mercaptoethanol and then denatured at 100°C for 10 minutes. Samples were separated on 8% to 12% sodium dodecyl sulfate polyacrylamide gels and transferred to PVDF membranes (Millipore). Afterwards, membranes were incubated in TBST containing 5% nonfat milk for 2 hours at room temperature. Next, membranes were incubated according to instructions with the respective primary antibodies overnight at 4°C and then treated with the appropriate horseradish peroxidase (HRP)–conjugated secondary antibodies (1:3000 dilutions). The blots were developed with ECL Western blotting reagents (Millipore). HRP-conjugated anti-mouse and anti-rabbit antibodies were purchased from Cell Signaling Technology (Danvers, MA). The following antibodies were used: SIRT1 (sc15404, Santa Cruz), SIRT2 (s8447, Sigma), SIRT3 (s4072, Sigma), β-Catenin (ab32572, Abcam), E-Cadherin (3195s, CST), PEPCK1 (sc32879, Santa Cruz), JNK (9258, CST), p-JNK (4668, CST), ERK (4695, CST), p-ERK (4370, CST), RAS (3965, CST), MMP-9 (ab137867, Abcam), actin (A5441, Sigma), Tubulin (2144, CST), Ac-Tubulin (5335, CST), anti-mouse IgG (7076, CST), and anti-rabbit IgG (7074, CST).
4
Western Blot Analysis of Sirt3, Acetyl Lysine, and Coq2
5
Quantitative Protein Analysis Protocol
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Whole cell lysates were collected using RIPA buffer with added protease inhibitor cocktail (Roche). Insoluble fractions were collected using Urea-containing buffer. Protein was quantified using Bradford assay reagent (Biorad) and bovine serum albumin standards (Pierce). Equal amounts of protein were electrophoresed in SDS-PAGE gels and transferred to PVDF membrane Immobilon-FL (Millipore). Blots were developed using ECL-plus (GE) for 5 minutes, chemiluminescence visualized on a Licor Odyssey FL imager and quantitation done using Licor Image Studio software. All quantitated protein signals were normalized to GAPDH signal from the same gel. Antibodies used were as follows: SIRT1 (Cell Signaling), SIRT2 (Abcam), SIRT3 (Sigma), Involucrin (Santa Cruz), Cytokeratin 10(Santa Cruz), GAPDH (Santa Cruz), p-NBS1 Ser 343 (Cell Signaling), NBS1 (Santa Cruz), ac-p53 (Cell Signaling), p53 (Calbiochem), pATM Ser 1981 (Cell Signaling), ATM(Cell Signaling), pCHK2 Ser 19 (Cell Signaling), CHK2(Cell Signaling).
6
Western Blot Analysis of Metabolic Proteins
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The cells were lysed using lysis buffer (Biosharp) to obtain total protein. Next, loading buffer containing 5% 2‐mercaptoethanol was mixed with the lysate. The proteins were denatured at 100°C for 10 minutes before being separated on 8%‐12% sodium dodecyl sulfate‐polyacrylamide gels. After transfer to PVDF membranes (Millipore), the membranes were incubated for 2 hours at room temperature in TBST containing 5% skim milk. Afterward, membranes were incubated with the corresponding primary antibodies overnight at 4°C according to the manufacturer's instructions. After treatment with the appropriate horseradish peroxidase (HRP)‐conjugated secondary antibodies, the membranes were incubated with ECL western blotting reagents (Millipore). The following antibodies were used: SIRT3 (s4072; Sigma), cMYC (ab32072; Abcam), HIF1α (36169; CST, Danvers, MA, USA), PDK1 (5662; CST), p‐PDHA1 (ab92696; Abcam), GAPDH (ab128915; Abcam), Actin (A5441; Sigma), PDHA1 (ab110334; Abcam), GLUT1 (ab115730; Abcam), Anti‐mouse IgG (7076; CST), and Anti‐rabbit IgG (7074; CST).
7
Immunohistochemical Analysis of Kidney Proteins
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Immunohistochemical staining was performed on formalin-fixed and paraffin-embedded 5 μm kidney sections. Following deparaffinization and rehydration, the slides were subjected to heat-mediated antigen retrieval in citrate buffer pH 6 and blocked with 3% BSA. The sections were probed with Sirt1 (Abcam catalogue # ab110304), Sirt3 (Sigma catalogue # S4072) or STING (Cell Signaling, catalogue # 13647S) antibody and incubated at room temperature for 1.5 h. Mouse/Rabbit PolyDetector reagent (Bio SB, Catalog No. BSB 0269) or UnoVue HRP secondary antibody detection reagent (Diagnostic BioSystems) was applied followed by DAB chromogen. The Periodic Acid-Schiff (PAS) staining was performed with a PAS stain kit (Thermo Scientific, Catalog No. 87007). Imaging was done with Nanozoomer (Hamamatsu Photonics) and Motic Digital Slide Scanner.
8
Western Blot Analysis of Mitochondrial Proteins
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Cells, washed with PBS, or pulverized flash frozen tissue was lysed in cold NP-40 lysis buffer with protease inhibitors (50 mM Tris, pH 7.5, 250 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 50 mM NaF, 0.2 mM Na3 VO4, 1 g/ml leupeptin, 1 g/ml pepstatin, 100 g/ml phenylmethylsulfonyl fluoride), sonicated for 5 s at 20% amplitude, and centrifuged at 14,000 rpm for 20 minutes at 4°C. Protein concentrations of lysates were assayed using the Bradford method (Bio-Rad Protein Assay). Equal amounts of protein were separated by SDS-PAGE electrophoresis and transferred to nitrocellulose membrane (GE Healthcare). Following blocking, membranes were probed with the following primary antibodies overnight at 4°C: SIRT3 (Cell Signaling 2627S), SIRT3 (EMD Millipore 07-1596), HSP60 (BD Transduction 611563), ATF5 (abcam ab184923), NRF1 (abcam ab55744), ClpP (abcam ab 124822), SOD1 (Santa Cruz sc-11407), FOXO3a (Cell Signaling 2497S), Actin (EMB Millipore MAB1501R), SOD2 (EMB Millipore 06-984), LC3 (MBL International PM036), SDHA (abcam ab14715), MTCO1 (abcam ab45918), GFP (Santa Cruz sc-9996), NRF2 (cell Signaling 12721). Blots were then probed with horseradish peroxidase conjugated anti-mouse (Jackson ImmunoResearch or KwikQuant) or anti-rabbit secondary antibodies (Thermo Fisher Scientific or KwikQuant) and detected using enhanced chemiluminescence (GE Healthcare or KwikQuant).
9
Western Blot Analysis of Metabolic Regulators
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Cells (5000 cells/μl) were lysed in 3x Laemmli-buffer. To disrupt all cells, the suspension was heated for 10 min at 95°C followed by centrifugation at 8,000 x g.
10 μl (~20 μg) of the cell lysate were loaded on 10%-SDS-PAGE and separated. Transfer to a nitrocellulose membrane was performed with semi-dry blot (Biometra, DE) at 200 mA for 20 min. We used primary antibodies for SIRT1 (Merck Millipore, USA), SIRT3 (Merck Millipore, USA), SIRT4 (Thermo Scientific, DE), AMPKα1 (Merck Millipore, USA), PPARγ (Merck Millipore, USA), PGC1α (Merck Millipore, USA) and β-Actin (Cell Signalling, DE) as a marker. For detection, we used the Odyssey FC system (LI-COR Bioscience, USA) with appropriate secondary antibodies.
10 μl (~20 μg) of the cell lysate were loaded on 10%-SDS-PAGE and separated. Transfer to a nitrocellulose membrane was performed with semi-dry blot (Biometra, DE) at 200 mA for 20 min. We used primary antibodies for SIRT1 (Merck Millipore, USA), SIRT3 (Merck Millipore, USA), SIRT4 (Thermo Scientific, DE), AMPKα1 (Merck Millipore, USA), PPARγ (Merck Millipore, USA), PGC1α (Merck Millipore, USA) and β-Actin (Cell Signalling, DE) as a marker. For detection, we used the Odyssey FC system (LI-COR Bioscience, USA) with appropriate secondary antibodies.
10
Western Blot Analysis of Mitochondrial Proteins
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