TWIST1 enhances the association of RELA with the IL8 promoter. (A) Fractionation-coupled Co-IP experiments (upper panel) with BT549 cell lysates using α-TWIST1 antibodies or IgG control. Cyto or c, cytoplasmic; n, nuclear. (B) ChIP assays using α-TWIST1 antibodies with solublized chromatin collected from MCF10A cells transfected with pcDNA, wild type TWIST1, S144R K145E or DWR TWIST1 mutant for 48 h. (C) ChIP assays with MCF10A and MCF10ATw cell lysates (left), or lysates from BT549 cells transduced with shCtrl or shTw (right), using α-RELA antibodies and IgG control. (D) ChIP assays with BT549 cell lysates using α-TWIST1 (left) or α-RELA antibodies and IgG controls. Mean ± SD, n = 3. IL8p, IL8 promoter amplified with primers flanking the E-box and kB binding sites (Figure 2A); IL8pCtrl#2, IL8 promoter enhancer region amplified with primers 2 kb upstream of transcription initiation site (primer sequences see Methods).