Secondary antibody conjugated to horseradish peroxidase

An ELISA-based assay was used to measure the NF-κB activity, as described previously [37 (link)–39 (link)]. In brief, after stimulation, cells were rinsed twice with cold PBS, and then RIPA containing a protease inhibitor cocktail (Roche, Basel, Switzerland) was added. After incubation on ice for 30 min, the lysate was centrifuged for 15 min at 14 000 rpm and the supernatant was collected. After being quantified with BCA reagent (Pierce, Rockford, IL), the cell extracts were incubated in a 96-well plate coated with the oligonucleotide containing the NF-κB consensus-binding site (5′-GGGACTTTCC-3′). Activated transcription factors from extracts specifically bound to the respective immobilized oligonucleotide. NF-κB activity was then detected with the primary antibody to NF-κB p65 (1 : 1000, Proteintech, China) and secondary antibody conjugated to horseradish peroxidase (1 : 10 1000, Abbkine, CA). Tetramethylbenzidine (100 μL, Sigma) was added in each microwell at 37°C before adding 100 μL of stopping solution (2M H₂SO₄). NF-κB activity was finally determined as absorbance values measured with a microplate reader at a wavelength of 450 nm.

NF-κB and Sp1 activity was assayed by an ELISA-based method as described previously [53 (link)]. Total cell extracts were collected and quantified with BCA reagent (Pierce, Rockford, IL). The cell extracts were incubated in a streptavidin-coated 96-well plate (Roche Diagnostics Corporation, Indianapolis, IN 46256), which had been coated with 3’end biotinylated oligonucleotide containing the conservative binding site NF-κB or Sp1. The sequences were shown in Supplementary Table 1. Activated transcription factors from extracts specifically bound to the respective immobilized oligonucleotide. NF-κB or Sp1 activity was detected with NF-κB or Sp1 antibody (1:500, Proteintech, China) and secondary antibody conjugated to horseradish peroxidase (1:10 000, Abbkine, CA). NF-κB and Sp1 activity was finally determined as absorbance values measured with a microplate reader at a wavelength of 450 nm.