The liver tissue and HepG2 cells were lysed by RIPA lysis solution (Beyotime, China) to extract the protein. The protein concentration was detected by BCA reaction kit. After quantitative analysis, the total protein was denatured in this study. SDS-Page gel was used for electrophoresis, electrophoresis apparatus (Bio-RAD, USA) was adjusted to 120 V for electrophoresis, PVDF membrane (Millipore, USA) was used for membrane transfer, and skim milk (Sigma, USA) for blocking. Primary antibodies (Abcam, UK): Sirt1 (1:1000; ab189494), optic atrophy 1 (Opa1, 1:1000; ab157457), mitofusin 2 (Mfn2, 1:1000; ab124773), Drp1 (1:1000; ab184247), NRF1 (1:1000; ab34682), mitochondrial transcription factor A (TFAM, 1:1000; ab252432; Abcam; UK), Bcl-2 (1:2000; ab182858), cleaved-caspase 3 (1:500; ab2302), BCL2-associated X protein (Bax, 1:1000; ab32503), cleaved-caspase 9 (1:2000; ab32539) and GAPDH (1:2500; ab9485) were then added overnight to incubate. The next day, goat anti-rabbit antibody (1:2000; ab288151) was incubated for 1 h with slow shaking at 25 °C. The immunoreactive bands were visualized by intensive chemiluminescent reagent. The gray value was analyzed by ImageJ software.
Ab189494
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab189494 is a lab equipment product manufactured by Abcam. It is a primary antibody used in various research applications. The core function of this product is to specifically bind to a target antigen, which can be used for detection and analysis purposes. Further details on the intended use or applications of this product are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (7 mentions)
Ab8227, by Abcam (4 mentions)
Ab205718, by Abcam (4 mentions)
Ab32503, by Abcam (3 mentions)
Ab9485, by Abcam (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Ab8245, by Abcam (3 mentions)
Ab16502, by Abcam (3 mentions)
Ab8226, by Abcam (3 mentions)
Ab184247, by Abcam (2 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Ab109012, by Abcam (2 mentions)
Ab196495, by Abcam (2 mentions)
Ab181602, by Abcam (2 mentions)
Ab52857, by Abcam (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Ab68477, by Abcam (2 mentions)
Ab62352, by Abcam (2 mentions)
Ab16048, by Abcam (2 mentions)
Ab133504, by Abcam (2 mentions)
54 protocols using ab189494
1
Mitochondrial Dynamics and Apoptosis Regulation in Liver Cells
2
Protein Analysis of Rat Myocardial Tissue
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A protein extraction kit (KeyGen Biotech., Ltd., Nanjing, China) was used to extract proteins from rat myocardial tissue homogenates, and the bicinchoninic acid method was applied to assess the protein concentrations. Equal amounts of extracted protein were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred on to a nitrocellulose membrane. The primary antibodies used in the experiment were: an anti-Bcl-2 antibody (1:1000, ab196495, Abcam), an anti-cleaved caspase-3 antibody (1:500, ab49822, Abcam), an anti-LC3B antibody (1:3000, ab51520, Abcam), an anti-Beclin-1 antibody (1:2000, ab207612, Abcam), an anti-p62 antibody (1:10000, ab109012, Abcam), an anti-SIRT1 antibody (1:500, ab189494, Abcam). An anti-GAPDH antibody (1:10000, ab181602, Abcam) was used as an internal control. The antigen–antibody complexes were observed by an enhanced chemiluminescence system (32106, Thermo, Rockford, IL, U.S.A.). The density of each protein band was quantified with Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, U.S.A.).
3
Analyzing Oxidative Stress Markers in MC3T3-E1 Cells
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Whole cell extracts of MC3TE-E1 for western blotting were prepared after the indicated treatment for 3 days as previously described [19 (link)]. 20 μg protein from total cell lysate was prepared using PRO-PREPTM protein extraction solution (Boca Scientific Inc., Boca Raton, FL) and carried out subsequent electrophoresis, according to the manufacturer’s instruction. The primary antibodies against the following proteins: FoxO1 (Abcam, ab52857, 1:1000), SIRT1 (Abcam, ab189494, 1:1000), human catalase (CAT, Abcam, ab130029, 1:1000), glutathione peroxidase (GPX1, Abcam, ab108427, 1:1000), superoxide dismutase 2 (SOD2, Abcam, ab252426, 1:1000). Expression levels of the target protein were normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Boster, Wuhan, China, 1:2000) levels in each sample. The following day, HRP-conjugated goat anti-rabbit, which used as secondary antibodies, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and the results were detection and analysis by using an iBrightCL1000 (Invitrogen, Carlsbad, CA).
4
Protein Expression Analysis in Rat Testes
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The testes of rats were lysed in RIPA buffer with 0.1% cocktail and 10% phosphotransferase inhibitor. Total protein from each sample was subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis and subsequently transferred to nitrocellulose membranes (BIO-RAD, Hercules, CA, USA, 0.22 μM). Transferred membranes were blocked with 5% non-fat milk containing TBST (TBS containing 0.1% Tween-20) for 45 min at room temperature. Thereafter, primary antibodies including SIRT1 (1:1000 dilution, ab189494, Abcam, Cambridge, UK), HIF-1α (1:1000 dilution, ab228649, Abcam, Cambridge, UK) and β-actin (1:1000 dilution, ab8227, Abcam, Cambridge, UK) were respectively incubated overnight at 4 °C and three washing were performed before the incubation of horseradish peroxidase-conjugated goat anti-rabbit IgG (secondary antibody, 1:1500 dilution, sc-2004; Santa Cruz Biotechnology, CA, USA) for 30 min at room temperature. Finally, Bio-Rad ChemiDoc system was used to analyze the intensities of autoradiographic bands.
5
Quantifying SIRT1 Protein Expression
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After H
2O
2 stimulation or transfection, the cells were collected, and total proteins were extracted using RIPA buffer and quantified using BCA protein quantitative kit (Beyotime, Haimen, China). The protein samples were separated by SDS-PAGE and transferred onto PVDF membrane (Millipore, Billerica, USA). The membrane was probed with primary antibodies against SIRT1 (ab189494; Abcam, Cambridge, USA), followed by incubation with the corresponding HRP-conjugated secondary antibody. The protein bands were finally visualized using Immobilon Western Chemiluminescent HRP substrate (Millipore), and quantified using Quantity One software (Bio-Rad Laboratories, Hercules, USA). β-Actin was used as the loading control.
2O
2 stimulation or transfection, the cells were collected, and total proteins were extracted using RIPA buffer and quantified using BCA protein quantitative kit (Beyotime, Haimen, China). The protein samples were separated by SDS-PAGE and transferred onto PVDF membrane (Millipore, Billerica, USA). The membrane was probed with primary antibodies against SIRT1 (ab189494; Abcam, Cambridge, USA), followed by incubation with the corresponding HRP-conjugated secondary antibody. The protein bands were finally visualized using Immobilon Western Chemiluminescent HRP substrate (Millipore), and quantified using Quantity One software (Bio-Rad Laboratories, Hercules, USA). β-Actin was used as the loading control.
6
Quantitative Western Blot Analysis
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Total proteins were extracted from PC12 cells and brain tissues of mice by RIPA buffer (Beyotime). Equal proteins of each sample were separated by 10% sodium dodecyl sulphate-polyacrylamide gels (SDS-PAGE). Next, separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Billerica, MA, USA) and blocked with 5% non-fat milk for 1 h. Then protein samples were incubated with the following primary antibodies overnight at 4°C: anti-SIRT1 (1:1000, ab189494, Abcam) and anti-β-actin (1:6000, ab115777, Abcam). After the membranes were washed with tris buffered saline Tween (TBST), the horseradish-conjugated secondary antibody (1:10,000, ab205718, Abcam) was added to incubate at 37°C for 2 h. The immune-reactivity was visualized with a Chemiluminescence Detection Kit (Thermo Fisher Scientific, Shanghai, China) and the relative protein expression of SIRT1 over the β-actin was quantified by Gel-pro Analyzer software (Media Cybernetics, Maryland, USA).
7
Protein isolation and Western blot analysis
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Total protein was obtained from frozen heart tissue and H9c2 cells, the cytosolic and nuclear proteins were isolated using a commercial kit (78833, Thermo Fisher Scientific, United States) as described in our previous study (Hu et al., 2021b (link)). Then, total protein was loaded into 10% SDS-PAGE gels, electro-transferred to an Immobilon-P membrane (IPVH00010, Millipore, China) and incubated with the primary antibodies against cTnI (ab52862, Abcam, United States), p-cTnI (ab58546, Abcam, United States), SOD1 (ab13498, Abcam, United States), SOD2 (ab13533, Abcam, United States), Sirt1 (ab189494, Abcam, United States), p-Nrf2 (ab76026, Abcam, United States), Nrf2 (ab62352, Abcam, United States), heme oxygenase-1 (HO-1, ab68477, Abcam, United States), Lamin B1 (ab16048, Abcam, United States) and GAPDH (ab8245, Abcam, United States). After incubation with the secondary antibody, the membranes were scanned using an Odyssey infrared imaging system (LI-COR, United States), and GAPDH was selected as the internal control.
8
Renal Protein Expression Analysis
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Renal tissues and cells were lysed using RIPA lysis fluid. The protein concentration was determined using a BCA protein assay kit (P0010S, Beyotime, China). Equal amounts of protein were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk and incubated with one of the following primary antibodies: anti-Cleaved caspase-3 (9661/R, 1:1000, Cell Signaling Technology, United States), anti-SIRT1(ab189494, 1:1000, Abcam, UK), anti-PGC-1α(ab191838, 1:1000, Abcam, UK), anti-NRF1(ab175932, 1:1000, Abcam, UK), anti-TFAM(A13552, 1:1000, Abclonal, China), anti-MFN1(13798-1-AP, 1:1000, Proteintech, China), anti-DRP1 (ab184247, 1:1000, Abcam, UK), anti-p66Shc (ab33770, 1:1000, Abcam, UK), anti-cytochrome C(CytoC) (ab133504, 1:5000, Abcam, UK), anti- DIABLO (ab32023, 1:1000, Abcam, UK), overnight at 4°C. After washing with TBST, the PVDF membranes were incubated with a secondary antibody at 37°C for 1 h. The protein bands on the PVDF membranes were observed.
9
Western Blot Analysis of Macrophage Proteins
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Macrophages from each group were collected and lysed using RIPA lysis buffer, followed by protein quantification utilizing the bicinchoninic acid method. 10 μg total protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for separation and moved to the polyvinylidene difluoride (PVDF) membrane. 5% defatted milk powder in 0.5% TBST was prepared to block the membranes for 2 h. Primary antibodies against p-p65 (ab194926, 1: 1000, Abcam), p65 (ab16502, 1:2000, Abcam), SIRT1 (ab189494, 1:1000, Abcam), LOX-1 (ab214427, 1: 1000, Abcam), CD36 (GTX100642, 1:500, GeneTex, Shanghai) and GAPDH (GTX 100118, 1:10000; GeneTex) were employed to incubate the membrane at 4°C overnight. The membrane was rinsed 3 times by TBST and cultivated with Goat anti-rabbit secondary antibody (ab205718, 1:5000, Abcam) at room temperature for 2 h. The visualization and analysis of protein blots were carried out employing an ECL (Amersham) and Image J (version 1.52; National Institute of Health) [15 (link)].
10
Immunohistochemical Analysis of FOXO1, AKT, and Sirt1
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Immunohistochemical staining for FOXO1 phosphorylated at S256, AKT phosphorylated at S473, and Sirt1 was carried out using the avidin-biotin-peroxidase complex technique with antibodies against FOXO1 phosphorylated at S256 (Cell Signaling Technology, #84192), AKT phosphorylated at S473 (Cell Signaling Technology, #4060), and Sirt1 (Abcam, #ab189494).
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