Protein was extracted from frozen murine heart tissue or isolated cardiac myocytes using a Column Tissue and Cell Protein Extraction Kit (EpiZyme, China). BCA assay kits were used to measure the protein concentration. After boiling 10 min at 100 °C with loading buffer (EpiZyme, China), the protein samples were separated using 4–12% FuturePAGE™ gradient gels and MOPS-SDS running buffer (Nanjing ACE Biotechnology, China). The PVDF membrane (for iNOS detection, nitrocellulose membranes were used) was blocked with 5 % skimmed milk for 60–90 min, then incubated with primary antibody overnight at 4 °C. The secondary antibody was then added for 60 min at room temperature. An ECL regent (BioSharp, China) was used to visualize the protein bands using a gel imaging system (Bio-Rad Laboratories, Inc., USA). The following antibodies were used in the present study: iNOS (BD Transduction Laboratories™), Akt, nuclear factor erythroid 2–related factor 2 (Nrf2; Affinity Biosciences, China), phosphorylated (p)-Akt (Ser 473) (Wanleibio, China), NADPH oxidase 4 (NOX4), superoxide dismutase 2 (SOD2), p-Nrf2, translocase of outer mitochondrial membrane 20, heme oxygenase 1 (HO-1) (ProteinTech Group, Inc., China) and OXPHOS antibody cocktail (Abcam, UK).
Heme oxygenase 1 ho 1
Manufactured by Proteintech
Sourced in China
Heme oxygenase 1 (HO-1) is an enzyme that catalyzes the degradation of heme to carbon monoxide, biliverdin, and iron. HO-1 plays a critical role in cellular homeostasis and adaptive responses to oxidative stress.
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Lab products found in correlation
Image lab software, by Bio-Rad (2 mentions)
Loading buffer, by Ipsen (1 mentions)
Oxphos antibody cocktail, by Abcam (1 mentions)
Column tissue and cell protein extraction kit, by Ipsen (1 mentions)
P nrf2, by Proteintech (1 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
Coomassie brilliant blue g 250 kit, by Solarbio (1 mentions)
β actin, by Proteintech (1 mentions)
β actin, by Bio-Rad (1 mentions)
Caspase 3, by Proteintech (1 mentions)
Bcl 2, by Proteintech (1 mentions)
N acetyl l cysteine nac, by Selleck Chemicals (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg, by Proteintech (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Goat anti mouse igg, by Proteintech (1 mentions)
Lamin b1, by Proteintech (1 mentions)
Nad p h quinone oxidoreductase 1 nqo 1, by Abcam (1 mentions)
Gapdh, by Proteintech (1 mentions)
4 protocols using heme oxygenase 1 ho 1
1
Cardiac Protein Extraction and Western Blot Analysis
2
Protein Expression Analysis in Intestinal Tissues
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According to the instruction of manufacturer, total protein was extracted from intestinal tissues and IEC-6 cells, respectively. The supernatant was collected and quantified using a Coomassie Brilliant Blue G-250 kit (Solarbio, Beijing, China). 10% SDS-PAGE gel was used to separate the protein samples (25 μg), and the separated samples were transferred to PVDF membrane. 5% skimmed milk or 5% bovine serum albumin was diluted in Tris-buffered saline containing Tween (TBS-T) to seal the membranes (3 h, 37°C). And the sealed membranes were hatched overnight (4°C) with following primary antibodies: Bcl-2-associated X (Bax), B-cell lymphoma-2 (Bcl-2), cysteinyl aspartate specific proteinase 3 (caspase-3), SOD-2, Nrf2, heme oxygenase 1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), and β-actin (Proteintech, Wuhan, China). After the first antibody was incubated, the imprints were rinsed in TBS-T for three times and then were hatched (room temperature, 2 h) with a horseradish-conjugated goat anti-rabbit antibody (Proteintech, Wuhan, China). ECL detection system was used to detect protein abundance. Image Lab software (Bio-Rad, CA, USA) was used to measure protein quantitation in optical density units and standardized to the corresponding β-actin sample expression.
3
Investigating Cellular Responses to Oxidative Stress
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The following antibodies were purchased: primary antibodies to SIRT5 (Cell Signaling Technology, Danvers, MA), H2A histone family member X phosphorylated on S139 (γ-H2AX, Cell Signaling Technology), nuclear factor erythroid-2-related factor 2 (Nrf2; Proteintech, Wuhan, China), heme oxygenase 1 (HO-1; Proteintech), manganese-dependent superoxide dismutase (MnSOD)/SOD2 (Proteintech), breast cancer gene 1 (BRCA1, Cell Signaling Technology), histone H3 (Cell Signaling Technology), and β-actin (Cell Signaling Technology); and secondary antibodies, specifically goat anti-rabbit IgG (Proteintech), goat anti-mouse IgG (Proteintech), and tetramethylrhodamine (TRITC)-conjugated secondary antibody (Proteintech).
Cisplatin and ML385 (an Nrf2-specific inhibitor) were purchased from MCE, China. N-acetyl-L -cysteine (NAC), a reactive oxygen species (ROS) scavenger, was purchased from Selleck, China.
Cisplatin and ML385 (an Nrf2-specific inhibitor) were purchased from MCE, China. N-acetyl-
4
Radiation-Induced Nrf2 Pathway Activation
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IEC‐6 cells were plated in 6‐ well plates with 5 × 105 cells per well, incubated overnight and pretreated with 10a for 1 hour before 6.0 Gy of γ‐irradiation. After 24 hours of incubation, the cells were collected. To extract the total proteins, the cells were lysed with cold RIPA buffer supplemented with protease inhibitor cocktail and phenylmethylsulfonyl fluoride. And a Nuclear/Cytoplasmic Protein Extraction kit was used to extract the nuclear proteins. Equal amounts of proteins were resolved on a 10% SDS–PAGE gel, and the proteins were electroblotted onto polyvinylidene difluoride membranes. The membranes were blocked with TBST buffer containing 5% BSA and incubated for 1.5 hours at room temperature with antibodies recognizing Nrf2 (1:1000 dilution, Proteintech), NAD(P)H quinone oxidoreductase 1 (NQO1, 1:20 000 dilution, Abcam), heme oxygenase 1 (HO‐1, 1:3000 dilution, Proteintech), Bax (1:5000 dilution, Abcam), Bcl‐2 (1:1000 dilution, Abcam), Lamin B1 (1:5000 dilution, Proteintech) and GAPDH (1:20 000 dilution, Proteintech). The membranes were then incubated with the corresponding secondary antibodies for 1 hour. The protein bands were visualized using ECL chemiluminescence reagents, and their intensities were analysed using Image Lab™ software (Bio‐Rad).
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