Paraffin-section containing aortic tissue and PVAT were deparaffinized, dehydrated, and boiled for epitope retrieval using an antigen retrieval buffer at pH = 6.0 (Opal 4-color IHC Kit, PerkinElmer). Tissue sections were blocked (StartingBlock™ T20 Blocking Buffer, 37539; Thermo-Fisher Scientific) for 1 h and incubated with primary antibodies (cathepsin S: ab18822, Abcam; MMP-12: PA5-13181, Thermo-Fisher Scientific; F4/80: ab6640, Abcam; CISD1: 16,006–1-AP, Proteintech; and NF-κB p65: SC-372, Santa Cruz) overnight at 4℃. The sections were incubated with HRP-conjugated secondary antibodies for 1 h and administrated with 50 × diluted Opal working solution for 10 min at room temperature. The following primary antibodies were stained with a repeat procedure including antigen stripping and blocking steps. DAPI was used to stain cell nuclei via adding mounting media containing fluoroshield with DAPI. The images were visualized by confocal microscopy (C1-Si, Nikon).
Nf κb p65 sc 372
Manufactured by Santa Cruz Biotechnology
Sourced in United States
NF-κB p65 (sc-372) is an antibody product offered by Santa Cruz Biotechnology. It is designed to detect the p65 subunit of the NF-κB transcription factor complex.
Automatically generated - may contain errors
Lab products found in correlation
Phospho mek1 2, by Cell Signaling Technology (2 mentions)
P3200, by GenDEPOT (2 mentions)
West q pico dura ecl solution, by GenDEPOT (2 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Notch1, by Abcam (2 mentions)
Nrf2 c 20 sc 722, by Santa Cruz Biotechnology (2 mentions)
Corm 2 288144, by Merck Group (2 mentions)
N acetyl cysteine a7250 10g, by Merck Group (2 mentions)
Ferrous chloride fecl 2, by Sinopharm (2 mentions)
Diamidino 2 phenylindole dihydrochloride dapi c10005, by Beyotime (2 mentions)
Reactive oxygen species assay kit s0033, by Beyotime (2 mentions)
Annexin 5 fitc apoptosis detection kit c1062, by Beyotime (2 mentions)
Anti nqo1 antibody ab2346, by Abcam (2 mentions)
Corm 2, by Merck Group (2 mentions)
Ab18822, by Abcam (1 mentions)
Cisd1 16 006 1 ap, by Proteintech (1 mentions)
Ab6640, by Abcam (1 mentions)
Startingblock t20 blocking buffer, by Thermo Fisher Scientific (1 mentions)
Opal 4 color ihc kit, by PerkinElmer (1 mentions)
8 protocols using nf κb p65 sc 372
1
Multiplexed Imaging of Inflammatory Markers
2
Molecular Mechanisms in Cancer Cells
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Monoclonal antibodies against vascular endothelial growth factor (VEGF; sc-7269), caspase-3 (sc-7148), ERα (sc-73562), progesterone receptor (PR; sc-130071), Bax (sc-20067), Bcl-2 (sc-130308), cyclin D1 (sc-70899) and NF-κB p65 (sc-372) were provided by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). RPMI-1640 medium (Gibco-Invitrogen, Grand Island, NY, USA) was purchased from Shanghai Chemical Reagent Co. Ltd. (Shanghai, China). The tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma (Grand Island, NY, USA).
3
Western Blot Analysis of Liver Proteins
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Frozen mouse liver samples were homogenized and digested in 1× RIPA lysis buffer containing complete protease and phosphatase inhibitor cocktails (P3200; GenDEPOT, Barker, TX, USA). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were immunoblotted with antibodies against phospho-AKT (#4060; Cell Signaling Technology), phospho-MEK1/2 (#9154; Cell Signaling Technology), NFκB p65 (sc-372; Santa Cruz Biotechnology, Dallas, TX, USA), NOTCH1 (ab8925; Abcam), and GAPDH (#2118; Cell Signaling Technology). Finally, the immunoreactive proteins were detected using the West-Q Pico Dura ECL Solution (W3653; GenDEPOT, Barker, TX, USA).
4
Investigating Anti-Inflammatory Pathways
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Antibodies against iNOS (ab3523), Cox-2 (ab179800), HO-1 (ab13243), and IκB-α (ab32518) were purchased from Abcam (Cambridge, UK). NF-κB p65 (sc-372) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phosphorylated (p)-IKKα/β (#2697) was purchased from Cell Signaling Technology (Danvers, MA, USA). Nrf2 (bs-1074R) was purchased from Bioss Antibodies (Woburn, MA, USA). 5-ALA was provided by Neopharma Japan (Tokyo, Japan). SFC was obtained from Komatsuya Corporation (Osaka, Japan). LPS from Salmonella typhimurium and Pred were purchased from Sigma-Aldrich (St. Louis, MO, USA).
5
Immune Signaling Pathway Activation Assay
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Dimethyl sulfoxide was obtained from Thermo Fisher. Puromycin was obtained from Clontech and used at 3 μg/ml in cell culture medium. LPS and Polybrene were obtained from Sigma-Aldrich. Human recombinant IFN-β, IFN-γ, universal mammalian IFN, and TNF-α were obtained from PBL. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 were obtained from Gemini Bio-Products. Steady-Glo cell lysis/luciferin reagent was obtained from Promega. Poly(I-C) was obtained from GE Healthcare. Stocks of G10 were synthesized by the OHSU Medicinal Chemistry Core Facility. DMXAA was purchased from ApexBio. Antibodies against the following antigens were used (with the source indicated in parentheses): glyceraldehyde-3-phosphate dehydrogenase (GAPDH; sc-51906; Santa Cruz Biotechnology); IRF3 (sc-9082; Santa Cruz Biotechnology); human S386 phospho-IRF3 (catalog number 2562-1; Epitomics); STING (catalog number 3337; Cell Signaling); IPS-1 (A300-782A; Bethyl); IFIT1/ISG56 (PA3 848; Thermo Fisher); TRIF (4596; Cell Signaling); Mx1 (GTX111153; GeneTex); β-actin (MAB0501R; Thermo Fisher); IκBα (Santa Cruz sc-371; Santa Cruz Biotechnology); NF-κB P65 (sc-372; Santa Cruz Biotechnology).
6
CORM-2 and NAC Modulate Stem Cell Behavior
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CORM-2 (288144) and N-acetyl-cysteine (NAC) (A7250-10G) were purchased from Sigma-Aldrich (St.
Louis, MO, USA).CORM-2 was dissolved in dimethyl sulfoxide, and then diluted in culture media to achieve the required concentrations. Inactivated CORM-2 (iCORM-2) was prepared by dissolving CORM-2 under the same conditions for 3 days at room temperature to liberate all CO from the molecule [14]. NSC C17.2, a stable, fully characterized, mouse stem cell line [15] , was kindly provided by Prof. Jin WL (Institute of Nano Biomedicine and Engineering, Shanghai Jiao Tong University, Shanghai, China). Ferrous chloride (FeCl 2 ) was purchased from Sinopharm Chemical Reagent (Shanghai, China). Annexin V-FITC Apoptosis Detection Kit (C1062), Diamidino-2-phenylindole dihydrochloride (DAPI) (C10005) and Reactive Oxygen Species Assay Kit (S0033) were from Beyotime Biotechnology (Shanghai, China). The primary antibodies Nrf2 (C-20) (SC-722) and NF-κB P65 (SC-372) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Anti-NQO1 antibody (ab2346) was from Abcam (Cambridge, UK). ß-Actin (4970),Tublin(2148) and H3 (12167), were from Cell Signaling Technology (USA).
Louis, MO, USA).CORM-2 was dissolved in dimethyl sulfoxide, and then diluted in culture media to achieve the required concentrations. Inactivated CORM-2 (iCORM-2) was prepared by dissolving CORM-2 under the same conditions for 3 days at room temperature to liberate all CO from the molecule [14]. NSC C17.2, a stable, fully characterized, mouse stem cell line [15] , was kindly provided by Prof. Jin WL (Institute of Nano Biomedicine and Engineering, Shanghai Jiao Tong University, Shanghai, China). Ferrous chloride (FeCl 2 ) was purchased from Sinopharm Chemical Reagent (Shanghai, China). Annexin V-FITC Apoptosis Detection Kit (C1062), Diamidino-2-phenylindole dihydrochloride (DAPI) (C10005) and Reactive Oxygen Species Assay Kit (S0033) were from Beyotime Biotechnology (Shanghai, China). The primary antibodies Nrf2 (C-20) (SC-722) and NF-κB P65 (SC-372) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Anti-NQO1 antibody (ab2346) was from Abcam (Cambridge, UK). ß-Actin (4970),Tublin(2148) and H3 (12167), were from Cell Signaling Technology (USA).
7
CORM-2 and NAC Modulate Stem Cell Behavior
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CORM-2 (288144) and N-acetyl-cysteine (NAC) (A7250-10G) were purchased from Sigma-Aldrich (St.
Louis, MO, USA).CORM-2 was dissolved in dimethyl sulfoxide, and then diluted in culture media to achieve the required concentrations. Inactivated CORM-2 (iCORM-2) was prepared by dissolving CORM-2 under the same conditions for 3 days at room temperature to liberate all CO from the molecule [14]. NSC C17.2, a stable, fully characterized, mouse stem cell line [15] , was kindly provided by Prof. Jin WL (Institute of Nano Biomedicine and Engineering, Shanghai Jiao Tong University, Shanghai, China). Ferrous chloride (FeCl 2 ) was purchased from Sinopharm Chemical Reagent (Shanghai, China). Annexin V-FITC Apoptosis Detection Kit (C1062), Diamidino-2-phenylindole dihydrochloride (DAPI) (C10005) and Reactive Oxygen Species Assay Kit (S0033) were from Beyotime Biotechnology (Shanghai, China). The primary antibodies Nrf2 (C-20) (SC-722) and NF-κB P65 (SC-372) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Anti-NQO1 antibody (ab2346) was from Abcam (Cambridge, UK). ß-Actin (4970),Tublin(2148) and H3 (12167), were from Cell Signaling Technology (USA).
Louis, MO, USA).CORM-2 was dissolved in dimethyl sulfoxide, and then diluted in culture media to achieve the required concentrations. Inactivated CORM-2 (iCORM-2) was prepared by dissolving CORM-2 under the same conditions for 3 days at room temperature to liberate all CO from the molecule [14]. NSC C17.2, a stable, fully characterized, mouse stem cell line [15] , was kindly provided by Prof. Jin WL (Institute of Nano Biomedicine and Engineering, Shanghai Jiao Tong University, Shanghai, China). Ferrous chloride (FeCl 2 ) was purchased from Sinopharm Chemical Reagent (Shanghai, China). Annexin V-FITC Apoptosis Detection Kit (C1062), Diamidino-2-phenylindole dihydrochloride (DAPI) (C10005) and Reactive Oxygen Species Assay Kit (S0033) were from Beyotime Biotechnology (Shanghai, China). The primary antibodies Nrf2 (C-20) (SC-722) and NF-κB P65 (SC-372) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Anti-NQO1 antibody (ab2346) was from Abcam (Cambridge, UK). ß-Actin (4970),Tublin(2148) and H3 (12167), were from Cell Signaling Technology (USA).
8
Western Blot Analysis of Signaling Proteins
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Frozen mouse liver samples were homogenized and digested in 1× RIPA lysis buffer containing complete protease and phosphatase inhibitor cocktails (P3200; GenDEPOT, Barker, TX, USA). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene di uoride (PVDF) membrane. The membranes were immunoblotted with antibodies against phospho-AKT (#4060; Cell Signaling Technology), phospho-MEK1/2 (#9154; Cell Signaling Technology), NFκB p65 (sc-372; Santa Cruz Biotechnology, Dallas, TX, USA), Notch1 (ab8925; Abcam), and GAPDH (#2118; Cell Signaling Technology). Finally, the immunoreactive proteins were detected using the West-Q Pico Dura ECL Solution (W3653; GenDEPOT, Barker, TX, USA).
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