Increased DNA-binding and transcriptional activity of NFκB complexes expressed in TAM-resistant (MCF7/HER2, BT474) relative to TAM-sensitive (MCF7) breast cancer cells. A. Whole cell lysates were immunoblotted to compare endogenous protein expression of p50, p65, and bcl-3 components relative to β-actin. Aliquots were also immunoprecipitated (IP) with antibody to p50, and subsequently Western blotted (WB) to detect bcl-3 complexed to p50 relative to total immunoprecipitated p50. B. DNA-binding by individual NFκB p50 and p65 subunits from nuclear extracts, quantitated by ELISA-based Trans-AM™ assays (mean ± SE OD450nm values from triplicate samples). C. (NFκB)-luciferase reporter gene expression (mean ± SE values from triplicate samples) measured after transient co-transfection of cells with the firefly reporter plasmid and a Renilla luciferase vector for normalization, as described in Methods. Asterisks (*) indicate significant difference (p < 0.05) from control MCF7 values.