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 .
Nrf2 sc 722
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Nrf2 (sc-722) is a primary antibody that recognizes the Nrf2 (nuclear factor erythroid 2-related factor 2) protein. Nrf2 is a transcription factor that plays a key role in the regulation of genes involved in the antioxidant response and cellular defense mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
2 mercaptoethanol, by Bio-Rad (2 mentions)
β actin, by Merck Group (2 mentions)
P16 sc 468, by Santa Cruz Biotechnology (2 mentions)
Luciferase assay kit, by Promega (2 mentions)
Spa 896, by Stressgen (2 mentions)
Kas tf110, by Stressgen (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Hmox1, by Santa Cruz Biotechnology (1 mentions)
Hsp70, by Santa Cruz Biotechnology (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Lamin a c, by Merck Group (1 mentions)
Catalase, by Cell Signaling Technology (1 mentions)
Elastin, by Abcam (1 mentions)
Imagequant tl, by GE Healthcare (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
0.2 m nitrocellulose membrane, by GE Healthcare (1 mentions)
Benzonase nuclease, by Santa Cruz Biotechnology (1 mentions)
P0448, by Agilent Technologies (1 mentions)
16 protocols using nrf2 sc 722
1
Protein Extraction and Western Blot Analysis
2
Immunoblotting of Cellular Proteins
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Whole cell lysates for immunoblotting were prepared by dissolving cell pellets in Laemmli Sample Buffer containing 5% 2-mercaptoethanol (Bio-Rad). Antibodies used in this study are as follows: lamin A/C (MAB3211; MilliporeSigma, Burlington, MA), catalase (Cell Signaling Technology, Danvers, MA), Nrf2 (sc-722, Santa Cruz), p16 (sc-468; Santa Cruz), and β-actin (A3854; Sigma-Aldrich, USA).
3
Whole Cell Lysate Immunoblotting Protocol
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Whole cell lysates for immunoblotting were prepared by dissolving cells in Laemmli Sample Buffer containing 5% of 2-mercaptoethanol (Bio-Rad). Antibodies used for immunoblotting include p16 (sc-468, Santa Cruz), Nrf2 (sc-722, Santa Cruz), Elastin (Ab21607, Abcam), β-actin (A3854, Sigma-Aldrich).
4
Western Blot Analysis of NRF2 and GAPDH
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hCMs were washed with ice-cold PBS and lysed in TX-100 RIPA buffer with protease inhibitor (Halt™ Protease Inhibitor Cocktail (100×)), phosphatase inhibitor (Halt™ Phosphatase Inhibitor Cocktail (100×)) and benzonase nuclease (Santa Cruz Biotechnology). Protein concentrations were determined with Pierce BCA Protein Assay Kit (Thermo Scientific) and lysates were treated with 5× sample buffer (312.5 mM Tris, pH = 6.8, 50% glycerol, 0.37 mM bromphenol blue, 347 mM SDS, 2.5% β-mercaptoethanol). Equal protein amounts were separated by SDS-polyacrylamide gel electrophoresis and transferred to 0.2-µM nitrocellulose membranes (GE Healthcare). Membranes were blocked in 5% BSA for 1 h. Primary antibodies (NRF2 sc-722, Santa Cruz, 1:500; GAPDH 14C10, Cell Signaling, 1:10,000) were diluted in blocking solution and incubated overnight (4 °C). HRP-conjugated secondary anti-rabbit antibodies (P0448, Dako, 1:5000) were incubated for 1 h at RT. ECL detection (Merck Millipore) was used for visualization with the Amersham Imager 600 (GE Healthcare). Band intensities were quantified in ImageQuant TL (GE Healthcare). The uncropped blots are depicted in the source data file.
5
Nrf2 Immunofluorescence in HepG2 Cells
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HepG2 cells were seeded on to 18 mm cover slips (ethanol and UV sterilized) in six well plates for 2–3 days and was treated as described before. Cells were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.25% Triton X-100 in PBS (pH 7.4). After blocking with 1% bovine serum albumin (BSA) in PBS for 30 min, they were incubated with Nrf2 (sc-722, Santa Cruz Biotechnology) at 1:500 dilution in PBS containing 1% BSA for overnight at 4°C. Nrf2 was detected with the secondary antibody conjugated with FITC (F0382; Sigma–Aldrich) and washed with PBS. The cells were counterstained with hoechst 33342 and mounted in Fluoroshield mounting medium onto a glass microscopic slide. Immunofluorescence analysis was performed with a laser scanning confocal imaging system coupled to a FV1000 Confocal Microscope (Olympus, Japan). Cells were viewed with a 63× objective lens (Numerical aperture 1.35), and images were captured with FluoView software (Olympus, Japan).
6
Cellular Oxidative Stress Assays
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β-actin (sc-130300) and Nrf2 (sc-722) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and the HO-1 (RT1270) antibody was purchased from Epitomics; Abcam (Cambridge, MA, USA). MTT and CoCl2 were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Lipofectamine® 2000, the intracellular superoxide probe dihydroethidium (DHE) and Rhodamine (Rho) 123 were all purchased from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA).
7
Western Blot Analysis of Cellular Protein Extracts
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Isolation of total and nuclear extracts was performed as described elsewhere [28] (link). Proteins (30 μg) were separated by SDS-PAGE on 10% acrylamide separation gels and transferred to Immobilon polyvinylidene difluoride membranes (Millipore). Western blot analysis was performed using antibodies against VLDLR (sc-18824), Nrf2 (sc-722), Nqo1 (sc-393736), ATF4 (sc-200) (Santa Cruz), VLDLR (AF2258) (R&D system), eIF2α (9722), phospho-eIF2α (Ser51) (9721), IgG control (2729S) (Cell Signaling Technology Inc., Danvers, MA), actin (A5441) (Sigma–Aldrich, Madrid, Spain). Detection was achieved using the Western Lightning® Plus-ECL chemiluminescence kit (PerkinElmer, Waltham, MA, USA). The equal loading of proteins was assessed by Ponceau S staining. The size of detected proteins was estimated using protein molecular-mass standards (Bio-Rad).
8
Protein Extraction and Western Blot Analysis
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Total proteins were extracted by RIPA lysis buffer which mixed with proteinase inhibitor (PMSF). The proteins were next separated by 12% SDS-PAGE and transfered to the PVDF membrane (Millipore). Then blocking for 2 h in 5% non-fat dry milk which was dissolved with 1 × Tris-buffered saline and incubated with the corresponding antibody [ATG5 (NB110–53818; NOVUS 1:500); ATG7 (MAB6608; R&D 1:500); LC3 (NB100–2220; NOVUS 1:1000); SQSTM1/p62 (ab56416; abcam 1:1000); Caspase-3 (#9662; CST 1:1000); Caspase-12 (ab62484; abcam 1:1000); PERK (sc-13,073; Santa Cruz 1:200); p-PERK (sc-32,577; Santa Cruz 1:200); Nrf2 (sc-722; Santa Cruz 1:500); XBP1 (sc-7160; Santa Cruz 1:200); ATF4 (#11815; CST 1:1000); IRE1 (NB100–2323; NOVUS 1:1000); β-actin (B1033; Biodragon 1:8000)] at 4 °C overnight. The next day, the HRP-conjugated goat anti-mouse or anti-rabbit IgG was added to incubate for 2 h after washing for thrice. At last, an enhanced chemiluminescence reagent (Beyo ECL Plus, Beyotime) was used to visualized the band.
9
Quercetin and Diquat-Induced Oxidative Stress
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Dulbecco’s modified Eagle’s F12 Ham medium (DMEM/F12) and fetal bovine serum (FBS) were obtained from Invitrogen (Carlsbad, CA, USA). Quercetin (#PHR1488) and diquat (#45422) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against PARP-1 (#9532), Caspase-3 (#9661S), and LC3A/B (#4108S) were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against Bcl-2 (sc-492), Bax (sc-493), and Nrf2 (sc-722) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Claudin-1 (#519000), Claudin-3 (#341700), Claudin-4 (#364800), Occludin (#404700), zonula occludens (ZO-1, #617300), ZO-2 (#389100), and ZO-3 (#364100) were obtained from Invitrogen (Carlsbad, CA, USA). Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (#A0208), HRP-labeled goat anti-mouse IgG (#A0216), JC-1 and EdU detection kits were purchased from Beyotime Biotechnology (Haimen, China). GSH detection kit was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Unless indicated, all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).
10
Western Blot Analysis of Protein Fractions
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