Transcription factor assay kit

Nuclear proteins were extracted from BV2 microglia or cerebral tissues per manufacturer’s protocol (Nuclear Extraction Kit, Panomics, Santa Clara, CA, USA). Protein concentration of nuclear extracts was measured using the BCA assay (Thermo Fisher Scientific, Grand Island, NY, USA). NF-κB DNA binding activity was assessed using a quantitative detection kit (Transcription Factor Assay Kit, Cayman Chemical, Ann Arbor, MI, USA). According to the manufacturer’s protocol, the 96-well plates were pre-coated with the specific double-stranded DNA sequence that contains the transcription factor NF-κB (p65) response element. Approximately 10 µg nuclear protein was incubated in the coated plate at RT for 1 h while rocking the plate gently at 150 rpm. After washing, NF-κB (p65)-specific primary antibody (1:100 dilution) was added, followed by horseradish peroxidase-labeled secondary antibody (1:100 dilution). The absorbance was read at 450 nm on a microplate reader.

VL-17A nuclei were purified using the Nuclear Extraction Kit (Origene, Rockville, MD) and nuclear protein was quantified by BCA assay. PPARα DNA-binding in 7 µg of nuclear protein was measured using the Transcription Factor Assay Kit (Cayman Chemical) according to manufacturer’s instructions.

The presence of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was determined in nuclear extracts isolated from macrophages exposed to asbestos and harvested at 1, 2, 4, 6, 8, and 12-h post asbestos exposure. Cytoplasmic and nuclear extracts were prepared using a commercially available nuclear extraction kit (Cayman Chemical, Ann Arbor, MI, USA). A transcription factor assay kit (Cayman Chemical, Ann Arbor, MI, USA) was used to detect nuclear Nrf2. The assay was performed according to the manufacturer’s protocol. The transcription factor assay kit utilizes a specific double-stranded DNA sequence containing the Nrf2 response element to bind to Nrf2 molecules present in the nuclear fraction. The presence of Nrf2 in the nucleus was detected by ELISA per the manufacturer’s instructions. The data are reported as the ratio of the absorbance at 450 nm to the protein concentration of the nuclear extract (µg).

The presence of nuclear factor E2-related factor 2 (Nrf2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 subunit was determined in nuclear extracts isolated from human mesothelial cells exposed to asbestos and harvested at 0, 2, 4, and 8 h post asbestos exposure. Cytoplasmic and nuclear extracts were prepared using a commercially available nuclear extraction kit (Cayman Chemical Company, Ann Arbor, MI, USA). Transcription factor assay kits (Cayman Chemical Company, Ann Arbor, MI, USA) were used to detect nuclear Nrf2 and NF-κB transcription factors, as previously described [17 (link),18 (link)]. The Transcription factor assay kits utilize a specific double-stranded DNA sequence containing the Nrf2 and NF-κB response element, respectively. The data are reported as the ratio of the absorbance at 450 nm (OD₄₅₀) to the nuclear extract protein concentration (μg). Absorbance measurements were obtained using a SpectraMax i3x Multi-Mode microplate reader (Molecular Devices, Sunnyvale, CA, USA).