Transcription factor assay kit

An ELISA-based DNA binding assay was used to determine the effects of nimbolide on the DNA binding of the transcription factors NF-κB and Nrf2. Nuclear extracts were obtained from BV-2 microglia treated with nimbolide (125, 250 and 500 nM) for 24 h. These extracts were then used for DNA binding assays using TransAM Nrf2 transcription factor ELISA Kit (Activ Motif, Belgium), containing a 96-well plate to which an oligonucleotide containing the antioxidant responsive element (ARE) consensus binding site (5′-GTCACAGTGACTCAGCAGAATCTG-3′) was immobilised.
Similarly, nuclear extracts were obtained from BV-2 microglia, which were treated with nimbolide (125, 250 and 500 nM) for 30 min prior to stimulation with LPS (100 ng/ml) for 1 h. Extracts were then used to evaluate DNA binding of NF-κB with a transcription factor assay kit (Abcam), which has a double-stranded DNA sequence containing the NF-κB response element (5′-GGGACTTTCC-3′) immobilised onto the bottom of the wells of a 96-well plate.

The systemic and local inflammation were assessed by testing the cytokines levels in homogenate and serum. The levels of tumour necrosis factor-α(TNF-α), Interleukin-1β (IL-1β) and Interleukin-10 (IL-10) in homogenate, as well as the levels of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemotactic protein 1 (MCP-1) in serum, were determined by the Enzyme-Linked Immunosorbent Assays with commercial kits (Wuhan Boster Bio-Engineering Limited Company, Wuhan, China) following the manufacturer’s protocols. The activity of NF-κB in lung tissues was measured using the Transcription Factor Assay Kit (Abcam, Toronto, Canada) according to the manufacturer’s instructions. The expression of NF-κB in lung tissue was determined with Western Blot.

To determine p65-NF-κB by ELISA, transcription Factor Assay Kit was used (ab133112, abcam). After the treatment of cells with LPS (10 μg/ml for 6 hrs), they were lysed with hypotonic HEPES lysis buffer (pH 7.4) and centrifuged at 1000 g for 10 min at 4°C, supernatants were collected and used for the determination of intracellular p65- NF-κB by ELISA as described earlier [30 (link)]. Briefly, a specific double stranded DNA (dsDNA) sequence containing the p65-NF-κB response element was immobilized onto the bottom of wells of a 96-well plate. p65-NF-κB contained in a nuclear extract, binds specifically to the p65-NF-κB response elements. p65-NF-κB was detected by addition of specific primary antibody directed against p65-NF-κB. A secondary antibody conjugated to HRP was added and the absorbance was read at 450 nm using spectrophotometer.

SW982 synovial cells were seeded on 60 mm culture dish overnight, then the cells were treated with LB (20 μM) for 1 h prior to treatment with IL-1β (20 ng/mL) for 2 h. Nuclear and cytoplasmic proteins in the cells were extracted following the procedures in the Transcription Factor Assay Kit (Abcam, Cambridge, UK), and NF-κB transcription factor binding activity of NF-kB (p65) was detected accordingly.

The effect of diannexin on inflammation was assessed by measuring BALF and serum cytokine levels. Intracellular adhesion molecule-1 (ICAM-1), macrophage inflammatory protein-1 (MIP-1), neutrophil elastase, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-8 were detected using ELISA kits (Wuhan Boster Bio-Engineering Limited Company, Wuhan, Hubei, China). We also counted the approximate number of neutrophils and macrophages in BALF deposits using Giemsa staining (Nikon 80i, Tokyo, Japan). Nuclear factor-κB (NF-κB) activity in the lung tissue was detected using the Transcription Factor Assay kit (Abcam, Toronto, Canada) according to the manufacturer's instructions. Briefly, nuclear proteins were extracted from lung tissues using the kit, and the extracted proteins were added into wells of the assay kit and incubated for 1 h before washing with wash buffer. The NF-κB antibody was then added and incubated with the extracted proteins in the wells for 1 h before washing with wash buffer and subsequent incubation with the HRP-conjugated secondary antibody. The secondary antibody was then washed off, and the reaction mixture was added and incubated for 30 min. NF-κB activity was analyzed using a microplate reader at 450 nm.

The systemic and local inflammation were assessed by testing the cytokines levels in homogenate and serum. The levels of tumour necrosis factor-α(TNF-α), Interleukin-1β (IL-1β) and Interleukin-10 (IL-10) in homogenate, as well as the levels of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemotactic protein 1 (MCP-1) in serum, were determined by the Enzyme-Linked Immunosorbent Assays with commercial kits (Wuhan Boster Bio-Engineering Limited Company, Wuhan, China) following the manufacturer’s protocols. The activity of NF-κB in lung tissues was measured using the Transcription Factor Assay Kit (Abcam, Toronto, Canada) according to the manufacturer’s instructions. The expression of NF-κB in lung tissue was determined with Western Blot.