Electrophoretic Mobility Shift Assay of NF-κB and AP-1 Binding

Electrophoretic mobility shift assay was performed as described previously (Guan et al., 2013 (link); Zhao et al., 2017 (link)). NF-κB and AP-1 DNA-binding activity was detected using a LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific, China). RAW264.7 cells were pretreated with taxifolin with a concentration of 100 μM for 2 h and subsequently stimulated with 50 ng/ml RANKL for 30 min. Nuclear extracts were prepared with Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology, Jiangsu, China). With help of LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific, China), an equal amount of nuclear extracts was incubated with biotin end-labeled duplex DNA and electrophoresed on a 6% polyacrylamide native gel. AP-1 and NF-κB probes (Beyotime Institute of Biotechnology, Jiangsu, China) used for EMSA containing the consensus recognition sites were as follows: NF-κB, 5′-AGTTGAGGGGACTTTCCCAGGC-3′; AP-1, 5′-CGCTTGATGACTCAGCCGGAA-3′.