Sp1 binds to the DNMT1 promoter. (A) Sp1 bound to putative Sp1-binding sites on DNMT1 promoter. EMSA assay of MV4-11 total extracts (T) or nuclear extracts (N) was performed using 3 different 32P-labeled DNMT1 promoter probes: DNMT1-Sp1-1 (abbreviated as D1); DNMT1-Sp1-2 (D2); and DNMT1-Sp1-3 (D3). Unlabeled oligos containing the consensus binding sites for Sp1 (Sp1) or unlabeled DNMT1 promoter probes (D) were added for competition with the 32P-labeled probes, thereby testing for DNA-binding specificity. (B) Antibody competition assays. Nuclear lysates were preincubated with Sp1 and NF-κB (p65) antibodies before formation of protein-DNA complex, thereby testing for protein-binding specificity. (C) Sp1 was physically associated with NF-κB and this association was disrupted by bortezomib. MV4-11 cells were treated with 60 nM bortezomib for 6 hours, and nuclear extracts were then applied to immunoprecipitation with NF-κB (p65) antibody. The immunocomplex was blotted with Sp1 antibody (top panel). Input controls (bottom panels). (D) Bortezomib inhibited NF-κB and Sp1 protein–binding activity. EMSA was performed with nuclear extracts prepared from MV4-11 cells untreated or treated with decitabine or bortezomib for 24 hours. DNA-consensus sequences containing Sp1- or NF-κB–binding sites were used as probes, shown on the top of each panel. C indicates untreated; D, decitabine (2.5 μM); and B, bortezomib (60 nM). Vertical lines have been inserted to indicate repositioned gel lanes.
