Transam nf κb

The DNA-binding capacity of NF-κB (p65 subunit) was measured in the nuclear extracts of HL60R cells treated using the TransAM™ NF-κB and Nuclear Extract™ Kits (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, the determination is based on a 96-well plate to which an oligonucleotide containing the NF-κB consensus binding site (5′-GGGACT TTCC-3′) has been immobilized. The activated NF-κB contained in extracts specifically binds to this nucleotide. By using an antibody that is directed against an epitope on p65 that is accessible only when NF-κB is bound to its target DNA, the NF-κB bound to the oligonucleotide is detected. The addition of a secondary antibody conjugated to horseradish peroxidase provides a sensitive colorimetric readout that is quantified by densitometry. The specificity of the assay is confirmed by contemporaneous incubations in presence of an excess of the non-immobilized consensus oligonucleotide, as a competitor, or of a mutated consensus oligonucleotide. The results were expressed as arbitrary units: one unit is the DNA binding capacity shown by 2.5 μg of whole-cell extract from Jurkat cells stimulated with 12-O-Tetradecanoylphorbol-13-acetate (TPA) and calcium ionophore (CI)/μg protein of nuclear extracts.

The DNA-binding capacity of NF-κB (p65 subunit) was defined in the nuclear extracts of SUM 149 and MDA-MB-231 cells using the TransAM NF-κB and Nuclear Extract kits (Active Motif, Carlsbad, CA, USA). The determination of binding capacity was based on a 96-well plate, on which an oligonucleotide containing the NF-κB consensus binding site was fixed. By use of an antibody directed against an epitope on p65, it may revealed NF-κB bound to the oligonucleotide. After addition of a horseradish peroxidase-conjugated secondary antibody, a sensitive colorimetric readout was quantified by densitometry (iMark Microplate Reader; Bio-Rad Laboratories, Inc.). The control of specificity of the assay carried out according to the indications of manufacturer’s protocol. The results were expressed as arbitrary units: one unit indicated the DNA binding capacity exerted by 2.5 μg whole cell extract from Jurkat cells/microgram of protein from the nuclear extracts. Jurkat cells, stimulated with 12-O-tetradecanoylphorbol-13-acetate and calcium ionophore, are the positive control for NF-κB p65 activation [35 (link)].

Tissues were homogenized in RIPA buffer and lysed for 30 min on ice. Samples were then sonicated, vortexed and centrifuged at 12,000 × g for 20 min at 4°C. Nuclear and cytoplasmic fractionation was performed using an EZ nuclei isolation kit (Applygen Technologies Inc.; China). Nuclear P65 concentrations and cytoplasmic IκB-α concentrations were determined using western blot analysis. Briefly, the supernatants were collected and separated using SDS-polyacrylamide gels, blotted onto membranes and incubated with the primary rabbit polyclonal anti-NF-κB p65 antibody, rabbit polyclonal anti-I-kappaB Kinase (IκB)-α antibody or ICAM-1 (M-19) antibody (Santa Cruz Biotechnology Inc., USA). Signals on the membranes were detected with an Odyssey Infrared Imaging System (LI-COR Bioscience, USA). The level of measured materials was normalized to the level of β-actin. Total lung NF-κB activity was determined using an activated NF-κB ELISA assay kit (TransAM NF-κB; Active Motif, Carlsbad, CA). In this kit, an oligonucleotide containing an NF-κB consensus site (5′-GGGACTTTCC-3′) was absorbed onto polystyrene microwells.

Concentrations of NF‐κB p65 in the nuclear extracts were measured. Nuclear extracts from in vitro experiments were prepared using a nuclear extract kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's protocol. The nuclear extracts were assayed using an ELISA kit (TransAM™ NF‐κB; Active Motif) to detect and quantify the NF‐κB activity according to the manufacturer's instructions. Briefly, 20 μg of the nuclear extract protein was incubated for 1 hour at 25°C in microwells coated with an oligonucleotide containing an NF‐κB p65 or p50‐binding consensus sequence. Next, the wells were incubated with rabbit anti‐NF‐κB p65 antibodies (1:1000 dilution) for 1 hour at 25°C, followed by incubation with peroxidase‐conjugated goat anti‐rabbit IgG (1:1000 dilution) for 1 hour at 25°C. Peroxidase activity was visualized by the tetramethylbenzidine reaction, and the optical density was measured at 450 nm.

To estimate NF-ΚB (p50 and p65 subunits) STAT3 and Nrf2 activation, enzymatic immunoassays with the Transcription Factor ELISA Kit (TransAM™NF-κB, TransAM™STAT3/TransAM™Nrf2, Active Motif, Carlsbad CA, USA) were used following the manufacturer’s protocol. Briefly, proper consensus-site double-strand oligonucleotides (NF-κB—5′-GGGACTTTCC-3′; STAT3-5’—TTCCCGGAA-3’; Nrf2-5ʹ—GTCACAGTGACTCAGCAGAATCTG-3ʹ) were immobilized on an ELISA plate and incubated for one hour with the nuclear extracts. The activated subunits bound to DNA were recognized with a specific primary antibody and detected with an HRP-conjugated secondary antibody. The number of subunits was determined by spectrophotometry at λ = 450 nm. The number of p50, and p65 subunits, STAT3, as well as the amount of Nrf2 contained in the DNA-binding complex, corresponded to the activated NF-κB, STAT3, or Nrf2 transcription factors.

The DNA binding capacity of NF-κB (p65 subunit) was measured in the nuclear extracts of cells treated using the TransAM NF-κB and Nuclear ExtractTM Kits (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. The results were expressed as arbitrary units: One unit is the DNA binding capacity shown by 2.5 µg of whole cell extract from Jurkat cells stimulated with 12-Otetradecanoylphorbol-13-acetate (TPA) + calcium ionophore (CI)/µg protein of HL-60/R nuclear extracts.