Gel-shift experiments with the ϵ^y-CACCC site and MEL cell nuclear extracts, with the use of SP1 and SP3 antibodies or a mutated CACCC site. (A) Shown are gel-shift experiments, with the radiolabeled 21-bp ϵ^y-CACCCwt double-stranded (DS) oligomer as probe. Lane 1 is probe only. Reactions in which probe was incubated with uninduced (lanes 2-5), DMSO-induced (lanes 6-9), and SCFA-induced (lanes 10-13), MEL cell nuclear extracts are shown. Lanes 3, 7, and 10 are incubations with a 200-fold excess of unlabeled probe as competitor. The addition of antibody raised against SP1 (Santa Cruz Biotechnology) to reactions (lanes 4, 8, and 12) causes a diminution of bands 1 and 2 (brackets), whereas antibody raised against SP3 (lanes 5, 9, and 13) causes a diminution in band 3 (asterisk). The supershifted complexes, unlabeled, are apparent in lanes 4, 5, 8, 9, 12, and 13. (B) Shown in lanes 1 through 7 and 8 through 14 are parallel gel shifts, in which binding to the radiolabeled 21-bp ϵ^y-CACCCwt DS oligomer at left is contrasted with binding to the mutated 21-bp ϵ^y-CACCCmut DS oligomer. Both probes were radiolabeled to a specific activity of greater than 1 × 10⁸. Probe-only lanes are 1 and 8; incubations with extracts from uninduced MEL cells are shown in lanes 2, 3, 9, and 10; from DMSO-induced cells in lanes 4, 5, 11, and 12; and from SCFA-induced cells in lanes 6, 7, 13, and 14. The results when 200-fold unlabeled cold-competitor is added are shown in lanes 3, 5, 7, 10, 12, and 14.