Cell culture dishes, plates, centrifuge tubes, and other plastic ware were purchased from BD Biosciences (Lincoln Park, NJ); Dulbecco modified Eagle medium (DMEM), Ham F-12, amphotericin B, and gentamicin were from Invitrogen (Grand Island, NY). Fetal bovine serum (FBS) was from Hyclone (Logan, UT). RNeasy Plus Mini RNA extraction kit from Qiagen (Valencia, CA); Ready-To-Go-Primer First-Strand Beads were from GE Healthcare (Piscataway, NJ); TaqMan gene expression assays and real-time PCR master mix were from Applied Biosystems (Foster City, CA). DCFDA—Cellular Reactive Oxygen Species Detection Assay Kit, rabbit polyclonal antibody against human malondialdehyde (MDA), 8-hydroxy-2-deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (HNE), aconitase-2, glutathione peroxidase-1 (GPX1), and mouse monoclonal antibody against superoxide dismutase-1 (SOD1) were purchased from Abcam (Cambridge, MA). Rabbit polyclonal antibody against human heme oxygenase-1 (HMOX1) and cyclooxygenase-2 (COX2) were from Santa Cruz Biotechnology (Santa Cruz, CA). β-actin antibody was from BioLegend (San Diego, CA). Fluorescein Alexa-Flour 488-conjugated secondary antibodies (donkey anti-rabbit, or goat anti-mouse IgG) were from Molecular Probes (Eugene, OR).
Hmox1
Manufactured by Santa Cruz Biotechnology
Sourced in United States
HMOX1 is a protein-coding gene that provides instructions for producing the enzyme heme oxygenase 1. This enzyme plays a crucial role in the metabolism of heme, a component of various proteins in the body.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (4 mentions)
Keap1, by Santa Cruz Biotechnology (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Cell culture dishes, by BD (1 mentions)
Centrifuge tube, by BD (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Glutathione peroxidase 1 gpx1, by Abcam (1 mentions)
Dcfda cellular reactive oxygen species detection assay kit, by Abcam (1 mentions)
Rneasy plus mini rna extraction kit, by Qiagen (1 mentions)
Ready to go primer first strand beads, by GE Healthcare (1 mentions)
Real time pcr master mix, by Thermo Fisher Scientific (1 mentions)
Cyclooxygenase 2 cox 2, by Santa Cruz Biotechnology (1 mentions)
β actin antibody, by BioLegend (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Nrf2 sc 722, by Santa Cruz Biotechnology (1 mentions)
Hsp70, by Santa Cruz Biotechnology (1 mentions)
Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
12 protocols using hmox1
1
Oxidative Stress Biomarkers Analysis
2
Protein Extraction and Western Blot Analysis
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Total protein was extracted from cells using radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher, Houston, TX, USA) containing 50 mM Tris–HCl, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, and 1% Triton X, plus Halt protease and phosphatase inhibitor (Thermo Fisher). Protein was quantified using Pierce BCA protein assay (Thermo Scientific) and read on a BioTek microplate reader at 570 nm. Protein was denatured by heating at 95 °C for 5 min and separated on a gradient SDS polyacrylamide gel electrophoresis gel (Thermo Fisher). The proteins were transferred onto a nitrocellulose membrane (LI-COR #926-31092, Lincoln, NE, USA), blocked using Odyssey blocking buffer (LI-COR #927-40000), incubated overnight with antibodies as follows, and visualized using a LI-COR Odyssey fluorescence scanner. The primary antibodies were Nrf2 (SC-722) 1:200 rabbit polyclonal (Santa Cruz Biotechnologies), GAPDH (MAB374) 1:1,000 mouse monoclonal (Chemicon Int., Temecula, CA, USA), HSP70 (SC 33575) 1:1,000 rabbit polyclonal (Santa Cruz Biotechnologies), tumor necrosis factor receptor–associated factor 6 (TRAF6; 04-451) 1:1,000 rabbit monoclonal (Chemicon), and HMOX-1 (SC10789) 1:100 rabbit polyclonal (Santa Cruz Biotechnologies), as shown in Table 2 .
3
Immunoblot Analysis of NRF2 and HMOX1
4
Protein Extraction and Western Blotting
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Total protein extracts were prepared from mouse liver or human HCC specimens by lysis with a solution comprising 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, aprotinin (10 µg/ml), leupeptin (10 µg/ml), 1 mM PMSF, 400 µM Na3VO4, 400 µM EDTA, 10 mM NaF, and 10 mM sodium pyrophosphate. The extracts (40 µg of protein) were fractionated by SDS-PAGE on a 10% gel, and the separated proteins were then transferred to a polyvinylidene difluoride membrane (IPVH00010; Millipore). The membrane was exposed to 3% dried skim milk and then incubated overnight at 4°C with primary antibodies to TfR1 (13–6800; Invitrogen), to Hmox1 (sc-10789; Santa Cruz Biotechnology), to Hsp90 (610419; BD Biosciences), to human IRP2 (sc-33682; Santa Cruz Biotechnology), or to mouse IRP2 (generated by K. Iwai, Kyoto University, Kyoto, Japan). Immune complexes were detected with HRP-conjugated secondary antibodies (Promega) and enhanced chemiluminescence reagents (Thermo Fisher Scientific; Nagahama et al., 2001 (link); Foster et al., 2003 (link); Hirano et al., 2013 (link)). For analysis of the human HCC specimens, the membrane was also stained with Coomassie Brilliant Blue as a loading control.
5
Antioxidant Effects of XXT on HUVECs
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Xueshuan Xinmaining Tablet (XXT) was provided by Jilin Huakang Pharmaceutical Co., Ltd. Human umbilical vein endothelial cells (HUVECs) were purchased from American Type Culture Collection (Manassas, VA, USA). TACS MTT Cell Proliferation Assay Kit and GPX enzyme-linked immunosorbent assay (ELISA) were purchased from R&D Systems (Minneapolis, MN, USA). Reactive Oxygen Species Assay Kit was obtained from Beyotime (Shanghai, China). Human Oxidative Stress PCR Array was purchased from Qiagen (Valencia, CA, USA). Anti-Nrf2, Keap1, GCLM, NQO1, HMOX1, and anti-Keap1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies were obtained from Sigma-Aldrich (St. Louis, MO, USA).
6
Western Blot Analysis of Protein Markers
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The cells were harvested with the 1X sample buffer (Biosesang, Seongnam, GG, Korea) containing a protease inhibitor and phosphatase inhibitor cocktail (GenDepot). Then, the total cell lysates were sonicated for 30 s and heated to 100 °C for 10 min. The proteins were separated on 8% polyacrylamide gels followed by transfer onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Burlington, MA, USA). ICAM-1 (Santa Cruz Biotechnology Inc.), HMOX-1 (Santa Cruz Biotechnology Inc.), phospho-β-catenin (Ser552; Cell Signaling Technology, Danvers, MA, USA), β-catenin (Santa Cruz Biotechnology Inc.), and β-actin (Santa Cruz Biotechnology Inc.) were used at 1/2000 dilutions.
7
Western Blotting of Xenograft Proteins
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For western immunoblotting experiments, tumor xenografts were lysed using the RIPA buffer supplemented with proteinase inhibitors (Bio-Rad Laboratories, Hercules, CA), which contained 5% 2-Mercaptoehanol (Sigma-Aldrich). The primary antibodies used were mouse monoclonal antibody γ-GCLM (sc-55586; Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal GCLC antibody (ab53179, Abcam, Cambridge, UK) and mouse monoclonal antibody HMOX1 (sc-136960, Santa Cruz Biotechnology). Anti-mouse IgG or anti-rabbit IgG secondary antibodies were purchased from Santa Cruz Biotechnology. A monoclonal mouse β-actin antibody (A5441, Sigma-Aldrich) was used as the loading control. Chemiluminescence images were obtained using ChemiDoc-MP Imaging system (ver 5.2.1, BioRad Laboratories Inc, Hercules, CA), and the band intensities were quantified using the Image J software ver. 1.52 (NIH, Bethesda, MD).
8
Western Blot Analysis of Key Proteins
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Briefly, for western blot analysis, 50 μg of proteins were loaded onto a 12% polyacrylamide gel Mini-PROTEAN® TGXTM (BIO-RAD, Milan, Italy). Electro-transfer to nitrocellulose membrane was obtained through Trans- Blot® TurboTM (BIO-RAD), using Trans-Blot® SE Semi-Dry Transfer Cell (BIO-RAD). Membranes were blocked in Odyssey Blocking Buffer (Licor, Milan, Italy), according to the manufacturer’s protocol. After blocking, membranes were washed three times in PBS for 5 min and incubated with primary antibodies against human α-SMA, TGF-β, HMOX1, TLR3, TLR4, E – Cadherin, Col1a, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), overnight at 4° C. The next day, membranes were washed three times in PBS for 5 min and incubated with Infrared anti-mouse IRDye800CW (1:5000) and anti-rabbit IRDye700CW secondary antibodies (1:5000) in PBS/0.5% Tween-20 for 1h at room temperature. All the antibodies were diluted in Odyssey Blocking Buffer. The obtained blots were visualized by Odyssey Infrared Imaging Scanner (Licor, Milan, Italy). Densitometric analysis was used for protein levels quantification, normalizing data to protein levels of β-actin.
9
Evaluating the NRF2-KEAP1 Signaling Pathway
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Protein samples were prepared either by RIPA buffer (R0278, Sigma-Aldrich) to yield total protein or by a nuclear extraction kit (Cat. #2900, Millipore Sigma) to yield cytoplasmic and nuclear proteins. The Criterion Vertical Electrophoresis Cell and Trans-Blot Turbo Transfer System (Bio-Rad) was used for immunoblot analysis as described in our previous study29 . Protein expression was detected by ECL Prime Western Blotting Detection Reagent (GE Healthcare Amersham). Primary antibodies used in the study were against NRF2 (#1272 T, Cell Signaling Technology), p-NRF2 Ser40 (#PA5-67520, Invitrogen), KEAP1 (#sc-514914, Santa Cruz Biotechnology), HMOX1 (#SC-136960, Santa Cruz Biotechnology), NQO1 (#sc-32793, Santa Cruz Biotechnology) and β-ACTIN (#SC-47778, Santa Cruz Biotechnology). HRP-linked anti-rabbit or anti-mouse IgG antibodies (#7074P2, #7076P2, Cell Signaling Technology) were used as secondary antibodies.
10
Protein Extraction and Western Blotting
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Total proteins were extracted from lung tissues and cells in a lysis buffer consisting of 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 5 mM β-glycerophosphate, and 1 μg/ml leupeptin. Comparable amounts of total protein (~40 μg) from each sample were separated and membranes were probed with antibodies specific for phospo-AKT (Cat # 2965), AKT (Cat # 4691) (Cell Signaling Technology, Baverly, MA), Hmox1 (Santa Cruz Biotech, Santa Cruz, CA), or Nqo1 (Abcam, Cambridge, UK). β-actin (Sigma, St. Louis, MO) was used as reference. The blots were developed using a HyGlo-ECL kit (Denville Scientific Inc, Metuchen, NJ).
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