BR is physically associated with EcR/USP in vitro. L57 cells were co-transfected with expression vectors for AaEcR-B, AaUSP-B and individual BR-V5 fusion proteins. Cell extracts were incubated with antibodies against EcR, and the precipitated proteins were subjected to Western-blot analysis with V5 antibody. At the bottom, AaEcR-B, AaUSP-B and AaBR proteins in the inputs were examined by mean of immunoblotting using antibodies against AaEcR, DmUSP and V5 tag, respectively. When the experiment was conducted using 20E, 1 × 10-6 M 20E was added into the binding and washing buffers.