Fig. 5.

Electrophoresis mobility shift assay and chromatin immunoprecipitation assay.

(a) Nuclear extracts (0.5 µg each) of Caco-2 (C) or DLD-1 (D) cells were mixed with 200 fmol of probes including a distal wild or mutated Sp1 site shown in Materials and Methods. EMSA was performed as described in Materials and Methods. Mutated oligo as shown in the Materials and Methods was used in some experiments. Cold oligo (x5 and x10) was used for the competition. (b) Supershift experiments using anti-Sp1 or anti-Sp3 antibody. Before mixing with a labeled probe, 2 µg/sample of non-specific antibody (control IgG), anti-Sp1 antibody or anti-Sp3 was added to nuclear extracts, and EMSA was performed. (c) ChIP assay was performed as described in Materials and Methods. Unrelated rabbit IgG, anti-Sp1 and anti-Sp3 antibodies were used to immunoprecipitate the DNA-protein complex. (–) denotes no antibody treatment. The size of the PCR product containing the distal Sp1 site was 251 bp.