Transam nf κb p65 assay kit

A total of 30 min after treatment, nuclear protein was isolated from the cells using a nuclear extract kit (Active Motif, Carlsbad, CA, USA), according to the manufacturer’s protocol. Protein concentrations of nuclear extract were determined using a protein assay kit. The DNA binding activity of NF-κB p65 was evaluated using a non-radioactive, ELISA-based TransAM NF-κB p65 assay kit (Active Motif, Carlsbad, CA, USA), according to the manufacturer’s protocol. The DNA binding activity of NF-κB p65 was normalized to the concentration of nuclear protein.

Total nuclear protein content was determined using a BSA-based protein quantification assay (ProStain; Active Motif). An ELISA-based transcription factor assay kit (TransAM NF-κB p65 Assay Kit; Active Motif) was used to quantify NF-κB p65 subunit nuclear DNA binding activity in pericytes and C₂C₁₂ cells from control and injured cocultures according to manufacturer's instructions. Briefly, 2 μg of nuclear protein was added to wells coated with a consensus binding sequence for NF-κB (5′-GGGACTTTCC-3′) and incubated for 1 h at room temperature. Wild type and mutated consensus oligonucleotides were used as competitors for NF-κB binding to ensure specificity of the reaction as per manufacturer's instructions. Wells were then washed, and a primary antibody directed at the p65 subunit was added and left to incubate for 1 h. This was followed by treatment of all wells with a secondary horseradish peroxidase-conjugated antibody. Then, developing solution was added to initiate a colorimetric reaction. After 5 min, a stop solution was added and absorbance was measured at 450 nm on a multiwell microplate reader (FLUOstar Optima; BMG Labtech, Offenburg, Germany). All samples were assayed in duplicate, and averages were used for data analysis.

NF-κB was determined in nuclear extracts from gastrocnemius muscle according to a modified protocol by Dignam et al.^{63 (link)} using an ELISA-based TransAM NF-κB p65 assay kit (Active Motif, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. Sample absorbance was read at 450 nm on a multiwell microplate reader (Spectra Max plus 284, Molecular Devices). Wild-type and mutated consensus oligonucleotides were used as competitors for NF-κB binding to ensure specificity of the reaction as per the manufacturer’s instructions. All the tests of the samples were run in duplicate, and the average value was used for data analysis.

The content of NF-κB binding to DNA in nuclear extracts was measured using a specific TransAM^® NF-κB p65 assay kit (Active motif, Carlsbad, CA, USA), according to the manufacturer’s instructions. Briefly, a 96-well plate was precoated with an oligonucleotide, containing the NF-κB p65 binding consensus site. The active form of the p65 subunit was detected using p65 antibodies, incubated for 1 h, specific for an epitope, which was accessible only when the appropriate subunit bound to its target DNA. An HRP-conjugated secondary antibody provided a colorimetric readout, which was quantified using a spectrophotometer (450 nm).

The activation of NF-κB binding to DNA was measured in nuclear extracts from synoviocytes pretreated with SP, CGRP and their inhibitors L-703606 (Sigma-Aldrich) and MK-3207 (Selleck Chemicals, Houston, TX, USA) with an ELISA-based TransAM NF-κB p65 assay kit (Active Motif). The NF-κB complex binding to the oligonucleotide was detected by spectrophotometry with the use of a secondary antibody conjugated to horseradish peroxidase. The results are presented as activation fold changes with the unstimulated control set to 100%.