Nf κb family transcription factor assay kit

Nuclear extracts were prepared and TransAM assays were performed as previously described.^[34] The activity of individual NF‐κB subunits was determined via ELISA using NF‐κB Family Transcription Factor Assay Kit (43296; Active Motif, Carlsbad, CA, USA). Briefly, nuclear extracts (2 µg) were placed into wells of NF‐κB consensus oligonucleotide‐coated 96‐well plates. Plates were incubated with NF‐κB primary antibody and then binding was detected using HRP‐conjugated secondary antibody included with the kit. For analysis, the optical density (OD) at 450 nm was measured using a Tecan Spark microplate reader (Tecan, Austria GmbH, Austria).

Preparation of nuclear extracts and TransAM assays were performed as previously described.^{55 (link)} The activity of individual NF-κB subunits was determined using an ELISA-based NF-κB Family Transcription Factor Assay Kit (43296; Active Motif, Carlsbad, CA, USA). Briefly, nuclear extracts (2 μg) were incubated in a 96-well plate, which was coated with NF-κB consensus oligonucleotides. The captured complexes were incubated with specific NF-κB primary Abs and subsequently detected using HRP-conjugated secondary Abs included with the kit. Finally, the optical density (OD) at 450 nm was measured using a Tecan Spark microplate reader (Tecan, Austria GmbH, Austria).

The activation of transcription factor NF-κB in U937 cells was determined using the NF-κB family-Transcription Factor Assay Kit (Active Motif, Carlsbad, CA, USA). Cells were plated at a concentration of 9×10⁶ cells and were harvested 18 h after M.F. infection and H₂S treatment. Cytoplasmic and nuclear cell fractions were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. Protein concentration was determined using Bradford protein assay method and equal amounts of protein extracts (20 μg for p52 and p65, 40 μg for p50) were analyzed. The activated NF-κB subunits were detected at 450 nm with a plate reader after treatment with primary antibodies directed against either NF-κB p65, p50 or p52 subunits and followed by a secondary antibody conjugated to horseradish peroxidase (HRP), according to the manufacturer’s instructions.