Nrf2 sc 365949

50 μg of protein from the homogenate was run on 12% SDS-PAGE (bis-acrylamide-laemmli gel) and blotted onto a polyvinylidene difluoride (PVDF) membrane (0.22 μm, Millipore, Billerica, MA, USA) and then blocked 1 h at room temperature with Tris buffer solution-0.01% Tween (TBS-T 0.01%) plus 5% nonfat milk. The membranes were incubated overnight at 4°C with mouse primary monoclonal antibodies eNOS (sc-376751), Nrf2 (sc-365949), Cu/Zn-SOD (sc-101523), and ORX (sc-398548) from Santa Cruz Biotechnology, Santa Cruz, CA, USA, with 1 : 1000 dilution. After that, the membranes were incubated overnight at 4°C with a secondary antibody that is conjugated with horseradish peroxidase with 1 : 10000 dilutions (Santa Cruz Biotechnology, Santa Cruz, CA, USA). All the blots were incubated with an α-actin antibody as a load control. The protein was detected by the chemiluminescence assay (Clarity Western ECL Substrate, Bio-Rad Laboratories, Inc., Hercules, CA, USA). Chemiluminescence that was emitted in this process was detected in X-ray films (AGFA, Ortho CP-GU, Agfa HealthCare NV, Belgium). Images from each film were acquired with a GS-800 densitometer (including Quantity One software from Bio-Rad). The values of the density of each band are expressed as arbitrary units (AU).

Fetal bovine serum (FBS), minimum essential medium (MEM), penicillin/streptomycin, trypsin-ethylene, and sodium pyruvate were purchased from Welgene (Daegu, Korea). Curcumin, B[a]P, phenylmethylsulfonyl fluoride, 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), formalin, isopropanol, dichlorofluorescein diacetate, oil red O (ORO), and Triton X-100 were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). The mounting medium for immunocytochemical analysis was purchased from Dako (Carpinteria, CA, USA). Antibodies against Nrf2 (sc-365949), AhR (sc-133088), CYP1A1 (sc-20772), CYP1B1 (sc-32882), NQO1 (sc-32793), m-IgGκ BP-HRP (sc-516102) and β-actin (sc-47778), siRNAs against Cyp1a1 and Cyp1b1 as well as the control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Catalase antibody (12980S), Anti-rabbit IgG HRP-linked Antibody (7074S), Anti-mouse IgG (H+L) F(ab’)₂ Fragment Alexa Flour 488 antibody (4408S) and Anti-mouse IgG (H+L), F(ab’)2 Fragment Alexa Flour 555 antibody (4409S) was purchased from Cell Signaling Technology (Danvers, MA, USA).

Liver protein extracts (50 μg) were run on 12% SDS-PAGE (bis-acrilamide-laemmli gel) and blotted onto a polyvinylidene difluoride (PVDF) membrane (0.22 μm Millipore, Billerica, MA, USA) and then blocked 1 h at room temperature with Tris buffer solution-0.01% Tween (TBS-T 0.01%) plus 5% non-fat milk. The membranes were incubated overnight at 4°C with mouse primary monoclonal antibody Nrf2 (sc-365949) from Santa Cruz Biotechnology, Santa Cruz, CA, USA, at a final dilution of 1:1000. After that, the membranes were incubated overnight at 4 °C with a secondary antibody that is conjugated with horseradish peroxidase and a dilution 1:10,000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). All of the blots were incubated with α-actin antibody as control. The protein was detected by chemiluminescence assay (Clarity Western ECL Substrate, Bio-Rad Laboratories, Inc., Hercules, CA, USA). Chemiluminescence that was emitted in this process was detected in X-ray films (AGFA, Ortho CP-GU, Agfa HealthCare NV, Belgium). Images from each film were acquired with a GS-800 densitometer (including Quantity One software from Bio-Rad). The values of the density of each band are expressed as arbitrary units (AU).

Protein extracts were obtained from either fresh or frozen plates 96 h after transfection using the protocol described for the enzyme activity assays. The protein level in each sample was quantified using the BCA assay according to the manufacturer’s protocol. Western blot analysis was carried out using 30 μg of protein, and after electrophoretic separation, proteins were transferred onto a PVDF membrane (Bio-Rad Laboratories, Feldkirchen, Germany). The membranes were then blocked with 0.5% of non-fat dry milk in 0.1% PBS-Tween, and then incubated with G6PD (ab993; Abcam, Cambridge, UK), NRF2 (sc-365949; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or β-actin (#69100; MP Biomedicals, Santa Ana, CA, USA) followed by exposure to corresponding anti-mouse (GR304350-1, Abcam, Cambridge, UK) or anti-rabbit (GR297013-4, Abcam, Cambridge, UK) horseradish peroxidase-conjugated secondary antibody. Visualization was carried out on Fujifilm X-ray (Fuji Medical X-ray Film, Dusseldorf, Germany) using chemiluminescence detection.

Protein was extracted from Huh7 cells and liver tissues. The isolated proteins were separated by polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes. Subsequently, the blots were exposed to primary antibodies against Kelch-like ECH-associated protein 1 (Keap1, #8047, CST, USA), nuclear factor (erythroid-derived 2)-like 2 (Nrf2, sc-365949, Santa Cruz, USA), heme oxygenase-1 (HO-1, #43966, CST, USA), and cleaved caspase-3 (#9661, CST, USA). Anti-GAPDH (TA-08), anti-β-tubulin (TA-10) and anti-β-actin (TA-09) were used as internal references, and were purchased from Beijing Zhong Shan-Golden Bridge Biological Technology Co., Ltd. The images of the gels were captured in a Bio-Rad Image Lab (ChemiDoc™ MP Imaging System, Bio-Rad, California, USA).

Glucose was obtained from Sigma (St. Louis, MO). ISL (PubChem CID: 638278) was sourced from Aladdin (Shanghai, China). The compound ISL dissolved in dimethyl sulfoxide and 0.5% sodium carboxyl methyl cellulose (CMC-Na) during in vitro and in vivo experiments, respectively. Antibodies against (TGF-β, sc-130348), Bcl-2-like protein 4 (Bax, sc-7480), B-cell lymphoma 2 (Bcl-2, sc-7382), extracellular signal-regulated kinase (ERK, sc-514302), phosphorylated ERK (p-ERK, sc-7383), Nrf-2 (sc-365949) and heme oxygenase-1 (HO-1, sc-136960) were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against c-Jun N-terminal kinases (JNK, 9252 S), phosphorylated JNK (p-JNK, 4668 S), P38 (9212 S), phosphorylated P38 (p-P38, 9211 S), Cl-C3 (cleaved caspase 3, 9664), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 5174 S) were obtained from Cell Signaling (Danvers, MA, USA). Antibodies against collagen type 4 (Col-IV, ab6586), TNF-α (ab1793), macrophage marker F4/80 (ab6640), and 3-nitrotyrosine (3-NT, ab61392) were obtained from Abcam (Cambridge, MA, USA). Horseradish peroxidase-conjugated anti-rabbit secondary antibodies were obtained from Santa Cruz Biotechnology (sc-2357). One-step TUNEL apoptosis assay kit was obtained from Beyotime (Beijing, China).