Binding of Sp1 to a non-canonical GC box downstream of the transcription start site.

(A) Overview of oligonucleotides used for EMSA. LKB1 DSE: downstream element, position +35 to +65 within the LKB1 5′-UTR and the corresponding mutant (LKB1 DSE Mut). Sequences of oligonuceotides containing consensus sites of different transcription factors used for competition are given below. (B) The radioactively labeled LKB1 DSE was incubated with 4 µg of nuclear extracts obtained from “444” cells (lane 1) or HepG2 cells (lane 2–9). A 500-fold molar excess of indicated unlabeled competitor oligos was added (lanes 3–9) and separated in a 7% polyacrylamide gel. (C) Complex formation (Sp1 S) of the same probe with 4 µg of nuclear extracts from “444” cells (lane 1) or from HepG2 cells (lane 2–6) was self-competed by a 500-fold molar excess of wild-type oligo (WT; lanes 3) but not by a mutant oligo (LKB1 DSE MUT; lane 4). Addition of an antibody against Sp1 (sc-59 X) (lane 5) retarded the specific complex (Sp1 SS) while addition of an antibody against Sp 3 (lane 6) did not.