Coimmunoprecipitation was carried out as described previously [36 (link)]. Briefly, bEnd.3 cells were subjected to OGD treatment and reperfusion, then lysed on ice in RIPA buffer. After preclearing with normal IgG, cell lysates (0.5 mg of protein) were incubated overnight at 4°C with 2 μg of anti-NOX4 (Proteintech, 1 : 100) and anti-p-STAT3 (CST, 1 : 100), followed by precipitation with 20 μL of protein A/G Plus-Agarose (Santa Cruz Biotech.) for 1 h at 4°C. The precipitated complexes were used for western blot analysis, as described below.
Anti nox4
Manufactured by Proteintech
Sourced in United States
Anti-NOX4 is a primary antibody that detects the Nox4 protein. Nox4 is a member of the NADPH oxidase (NOX) family of enzymes that produce reactive oxygen species. The Anti-NOX4 antibody can be used to identify and quantify Nox4 expression in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (3 mentions)
Anti nox2, by Proteintech (2 mentions)
Anti gapdh, by Proteintech (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Chemi lumi one super type, by Nacalai Tesque (1 mentions)
Image lab software version 6, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Anti lamin a c, by Cell Signaling Technology (1 mentions)
Hrp conjugated affinipure goat anti mouse igg, by Proteintech (1 mentions)
Anti sirt1, by Abcam (1 mentions)
Anti vwf, by Santa Cruz Biotechnology (1 mentions)
Selisistat, by MedChemExpress (1 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated affinipure goat anti rabbit igg, by Proteintech (1 mentions)
Anti lamin b1, by Proteintech (1 mentions)
Anti cd31 pecam 1, by Santa Cruz Biotechnology (1 mentions)
6 protocols using anti nox4
1
Co-Immunoprecipitation of NOX4 and p-STAT3
2
Western Blot Analysis of Cellular Proteins
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Western blot analysis was performed as previously described [47 (link)]. Total protein lysates of cells were obtained by lysis in RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% NP-40, 5 mM EDTA, 0.5% sodium deoxycholate, 20 mg/mL Na3VO4, 10 mM NaF, and 1 mM PMSF) with a protease inhibitor cocktail (04080-11; Nacalai Tesque, Kyoto, Japan). Total protein lysates (60 µg) from SH-SY5Y cells were subjected to gel electrophoresis and protein transfer onto a PVDF membrane (IPVH00010, Immobilon-P; Millipore, Burlington, MA, USA). The following primary antibodies were used: anti-β-actin (clone AC15; Sigma-Aldrich, St. Louis, MI, USA), anti-SIRT7 (clone D3K5A, #5360; Cell Signaling Technology, Danvers, MA, USA), anti-cleaved caspase 3 (#9661; Cell Signaling Technology), and anti-NOX4 (14347-1-AP; Proteintech). After reaction with secondary antibodies, the signals were detected by using Chemi-Lumi One Super Type (Nacalai Tesque) and a ChemiDoc imaging system (BR170-8265; Bio-Rad). All experiments were performed at least three times, and band intensities were quantified using Image Lab Software version 6.0 (Bio-Rad, Hercules, CA, USA).
3
Detailed Procedures for Cellular Senescence Assessment
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The reagents included carbon tetrachloride (Sigma‐Aldrich, 56‐23‐5), 30% H2O2 (Hydrogen peroxide 30%, Sigma‐Aldrich, 1.07298), selisistat (EX‐527, MedChemExpress, 49843‐98‐3), resveratrol (SRT501, MedChemExpress, 501‐36‐0), DAPI (Sigma‐Aldrich, D9542), Alexa Fluor™ 647 Phalloidin (Thermo, A22287), protease cocktails inhibitor (Beyotime, P1005) and PMSF (Phenylmethanesulphonyl fluoride, Beyotime, ST506).
The primary antibodies included anti‐α‐SMA (Boster, BM0002), anti‐vWF (Santa Cruz, SC‐365712), anti‐CD32b (ZEN‐bioscience, 382560), anti‐CD31 (PECAM‐1, Santa Cruz, sc‐18916), anti‐CD31 (Abcam, ab33858), anti‐NOX2 (Proteintech, 19013‐1‐AP), anti‐NOX4 (Proteintech, 14347‐1‐AP), anti‐Lamin A/C (Cell Signaling Technology, 4777S), anti‐Lamin B1 (Proteintech, 66095‐1‐Ig), anti‐progerin (Santa Cruz, sc‐81611), anti‐p53 (Abcam, ab131442), anti‐p53 (acetyl K381; Abcam, ab61241), anti‐SIRT1 (Abcam, ab110304), anti‐Histone H3 (Proteintech, 17168‐1‐AP) and anti‐GAPDH (Proteintech, 60004‐1). HRP‐conjugated Affinipure Goat Anti‐Mouse IgG (H + L; Proteintech, SA00001‐1), HRP‐conjugated Affinipure Goat Anti‐Rabbit IgG (H + L; Proteintech, SA00001‐2), FITC‐labelled goat anti‐rabbit IgG (H + L; Beyotime, a0562) and Cy3‐labelled goat anti‐mouse IgG (H + L; Beyotime, a0521) were used for secondary antibodies.
The primary antibodies included anti‐α‐SMA (Boster, BM0002), anti‐vWF (Santa Cruz, SC‐365712), anti‐CD32b (ZEN‐bioscience, 382560), anti‐CD31 (PECAM‐1, Santa Cruz, sc‐18916), anti‐CD31 (Abcam, ab33858), anti‐NOX2 (Proteintech, 19013‐1‐AP), anti‐NOX4 (Proteintech, 14347‐1‐AP), anti‐Lamin A/C (Cell Signaling Technology, 4777S), anti‐Lamin B1 (Proteintech, 66095‐1‐Ig), anti‐progerin (Santa Cruz, sc‐81611), anti‐p53 (Abcam, ab131442), anti‐p53 (acetyl K381; Abcam, ab61241), anti‐SIRT1 (Abcam, ab110304), anti‐Histone H3 (Proteintech, 17168‐1‐AP) and anti‐GAPDH (Proteintech, 60004‐1). HRP‐conjugated Affinipure Goat Anti‐Mouse IgG (H + L; Proteintech, SA00001‐1), HRP‐conjugated Affinipure Goat Anti‐Rabbit IgG (H + L; Proteintech, SA00001‐2), FITC‐labelled goat anti‐rabbit IgG (H + L; Beyotime, a0562) and Cy3‐labelled goat anti‐mouse IgG (H + L; Beyotime, a0521) were used for secondary antibodies.
4
Cell Lysis and Western Blot Analysis
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Whole cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche) as previously described (Liu et al., 2018 (link)). Western blot analyses were performed with anti-BRG1 (Santa Cruz, sc-10768), anti-collagen type I (Rockland, 600-401-103), anti-α-SMA (Sigma, A2547), anti-VE-Cadherin (Cell Signaling Technology, 2158), anti-PECAM1 (Proteintech, 11265-1), anti-NOX4 (Proteintech, 14347-1), and anti-β-actin (Sigma, A2228) antibodies.
5
Protein Expression Analysis in Vascular Cells
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Total protein was extracted from HAECs and vascular tissues derived from rats. Nuclear extracts were isolated from HAECs using a nuclear protein extraction kit (Beyotime, Shanghai, China). Protein samples were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk, the membranes were incubated with the appropriate primary antibodies including anti-TXNIP (1:1000; Proteintech), anti-TRX (1:2000; Proteintech), anti-AP1 (1:1000; Proteintech), anti-REF1 (1:1000; Bimake), anti-GAPDH (1:5000; Proteintech), anti-PCNA (1:2000; Proteintech), anti-NOX4 (1:1000; Proteintech), anti-NOX2 (1:1000; Santa Cruz Biotechnology), anti-SOD2 (1:2000; Santa Cruz Biotechnology), anti-eNOS (1:1000; Cell Signaling Technology), anti-p-eNOS (ser 1177) (1:1000; Cell Signaling Technology), anti-AKT (1:1000; Cell Signaling Technology), and anti-p-AKT (ser 473) (1:500; Santa Cruz Biotechnology) overnight at 4 °C. Then, the membranes were incubated with species-matched secondary antibodies (1:5000; Proteintech) for 1.5 h at room temperature. The blots were developed with a hypersensitive ECL chemiluminescence kit (Beyotime) and examined using a Bio-Rad gel imaging system (Bio-Rad, CA, USA).
6
Protein Extraction and Western Blot Analysis
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Whole-cell lysates were obtained by resuspending cell pellets in RIPA buffer (50 mmol/L Tris pH 7.4, 150 mmol/L NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche). Nuclear proteins were extracted essentially as described before. 23 (link) Antibodies were incubated with cell lysates overnight before being absorbed by Protein A/G-plus Agarose beads. Precipitated immune complex was released by boiling with 1X electrophoresis sample buffer. Western blot analyses were performed with anti-MKL1 (Santa Cruz), anti-MOF (Bethyl Laboratories), anti-NOX1, anti-NOX2, anti-NOX4 (Proteintech), and anti-β-actin (Sigma) antibodies.
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