Nuclear extract and NF-κB transbinding assay

A total of 1×106 cells/ml 4T1 cells were collected and nuclear extracts were isolated using the Nuclear Extract kit from Active Motif GmbH (Regensburg, Germany) according to the manufacturer's protocol. Nuclear extracts were stored at −80°C until they were used, and their concentration was measured using a Bradford assay. NF-κB DNA binding activity was determined using an Trans-AM P65-NF-κB ELISA-based kit (cat number 40096; Active Motif GmbH), which is a sensitive assay that measures the quantity of activated NF-κB in the nuclear extracts from the 4T1 breast cancer cell line, prior to and following Fmod and Dcn expression. In total, 10 µg nuclear extract was added to a biotinylated oligonucleotide containing the NF-κB consensus site attached to the streptavidin-coated 96-well plates. Plates were washed with wash buffer (cat number 40096; Active Motif GmbH) to remove all the unbound reagents; to visualize NF-κB DNA binding, an anti-p65 primary antibody (dilution, 1:2,000; cat number 40096; Active Motif GmbH) was added for 1 h at room temperature without agitation, followed by a goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (dilution, 1:5,000; cat number 40096; Active Motif GmbH) at room temperature without agitation. Subsequent to an incubation for 1 h at room temperature, 100 µl developing solution (cat number 40096; Active Motif GmbH) was added to all wells for 5 min at room temperature protected from direct light. The blue color development in the sample wells was monitored until it turned medium to dark blue. Subsequently, 100 µl stop solution (cat number 40096; Active Motif GmbH) was added and the blue color turned yellow. Finally, the absorbance value was ascertained using a spectrophotometer at a wavelength of 450 nm. For the p65 positive control: 2.5 µg of Jurkat nuclear extract was provided (1 µl of nuclear extract in 19 µl of complete lysis buffer per well according to the Active Motif kit protocol). For blank wells: 20 µl complete lysis buffer was added per well (according to the Active Motif kit protocol).