Figure 5.

Effect of phosphorylation/dephosphorylation of MutSα on GT-binding activity. (A) HeLa-MR cells were cultivated in phosphate-depleted medium and pretreated with the protein kinase inhibitor H7 or quercetin. Thereafter, phosphate-free medium was replaced with normal medium containing the protein kinase inhibitors and the cells were incubated for a further 2 h. Nuclear extracts were prepared and subjected to EMSA. The specific band is labelled by an arrow. The lower band appearing in all lanes is non-specific. (B) HeLa-MR cells were incubated for 2 h with 25 µM MNNG to increase the amount of nuclear MutSα and nuclear extracts for EMSA were prepared. Cell extract protein was incubated with CIP, λ-PPase or SAP or, as a control, with heat-denatured phosphatases (labelled i) or dephosphorylation buffer only and subjected to EMSA using a GC- (control) or GT-containing oligonucleotide. The specific binding complex is indicated by an arrow and the radioactively labelled substrate is marked by an asterik. (C) Recombinant MutSα was incubated with CIP or SAP or, as a control, with heat-inactivated CIP (labelled i) and subjected to EMSA using a GC- (control) or GT-containing oligonucleotide.