Methylation interference assay of the UP site. (a) Nuclear proteins were used in a bandshift assay with a UP probe that had been chemically methylated. The various complexes indicated in (c) were extracted, chemically cleaved, individually separated on a denaturing gel and autoradiographed. The G residues whose methylation blocks binding to the upper and lower complexes are shown in bold in the sequence of the UP site. Only the non-coding strand is shown. (b) Location of G residues sensitive to methylation. Methylation of six G residues, indicated by the arrows, block binding to the upper and lower complexes. A UP oligonucleotide (UPmut) with mutations at these residues (circled) was created. (c) Binding of nuclear proteins to wild-type and mutant UP probes. A bandshift assay was preformed with nuclear extracts using the wild-type UP oligonucleotide as a probe. The indicated amounts of cold wild-type UP or mutant UP oligonucleotides (oligo) were added into the reaction. The complexes correspond to Upper, Lower, non-specific (NS) and unbound (U).