Figure 5.

Binding activities of keratinocyte nuclear proteins to SP1-like sequences A) The sequence of the TGM1 promoter from -1540 to -1361 kb is shown. The overlapping underlined sequences labelled A-G were chosen as EMSA probes. Both strands were synthesized and annealed, leaving a single G 5' overhang to allow labelling with ³²P dCTP by a fill-in reaction. In some cases an additional G residue, not present in the gene sequence, was included at the 5' end of one strand. The T and A residues written above some bases in probes A and G indicate the base changes to make the mutants, TG-Amut and TG-Gmut. B) Probes were a consensus Sp1 site (Stratagene) or TGM1 promoter Sp1-like sites, TG-A and TG-G. Competitors were added as indicated or None for no competitor. To make the mutant consensus Sp1 site (Sp1mut), the core sequence GGGGCGGGG was changed to GGTTTTGGG. Nuclear extracts were from postconfluent SIK cells. C) Antibodies to Sp1, Sp3 or both were added as indicated to binding reactions with nuclear extracts from postconfluent SIK cells before addition of probes TG-A or TG-G. The no antibody control is indicated by None. An arrow shows a supershifted band visible after addition of Sp3 antibody.