Identification of Smad3 target genes by ChIP-chip in the MCF10A progression series. (A) The anti-Smad3 antibody recognizes a unique band in wild type and Smad2 null but not Smad3 null IMECs by Western blot. (B) ChIP-QPCR showing ability of Smad3 antibody (αS3) to immunoprecipitate Smad3 bound to the Smad7 promoter in Smad3 wild-type but not Smad3 knockout mouse embryo fibroblasts. CON, isotype-matched control antibody. (C) Time course of Smad3 occupancy at promoters of three previously characterized Smad3 target genes assessed by ChIP-QPCR following treatment of M3 cells with TGF-β. (D) Genome browser view (hg18) of Smad3 binding in the promoter of IFNK in M1 to M4 cells. The signal represents the difference between the TGF-β-treated and untreated conditions. The threshold represents signal intensity corresponding to FDR = 0.15. Black rectangles represent regions of significant Smad3 binding. (E) ChIP-QPCR validation of Smad3 occupancy at the IFNK locus. Results are mean +/−SD (n = 3) normalized to no TGF-β condition. *P <0.05 for enrichment >2-fold. (F) Enrichment of the canonical Smad binding element (SBE) in SBRs. The black line represents 190 high confidence SBRs and the grey line represents 190 random promoter regions with no Smad3 binding. The generic SMAD binding motif is shown. (G) Top 10 enriched transcription factor (TF) matrices within +/−250 bp of the center of 190 high confidence SBRs. (H) Schematic showing co-occurrence for the most enriched TF motifs. Pairwise analysis of each enriched motif was performed using the Fisher’s exact test with Bonferroni correction. The adjusted P values for co-occurrence of pairs of TFs are represented by the connecting lines: P <1e-7 (purple), P <1e-5 (pink), P <1e-2 (black). ChIP, chromatin immunoprecipitation; FDR, false discovery rate; IMEC, immortalized mouse mammary epithelial cells; QPCR, quantitative polymerase chain reaction; SBR, Smad binding region; TGF-β, transforming growth factor beta.