3.4  CK2-mediated phosphorylation of Sp1 is associated with decreased DNA binding
To further investigate the potential impact of CK2-mediated phosphorylation of Sp1 on DNA binding activity, experiments were carried out using purified CK2. Firstly, the effect of CK2-mediated phosphorylation of extracts from untreated macrophages on Sp1/Sp3 binding was analysed by EMSA. The probe used corresponded to the + 36/+ 90 region of the LPL gene [15] and contained all three Sp1/Sp3 binding sites. Consistent with our previous studies [9,15], three DNA-protein complexes were produced (C1 to C3; Fig. 4A). Antibody interference assays have shown that complex C1 is composed of Sp1 whereas complexes C2 and C3 consist mainly of Sp3 [9,15]. Phosphorylation of extracts from untreated cells with two different concentrations of CK2 reduced the binding of Sp1/Sp3 to levels observed in IFN-γ-treated cells (Fig. 4A). Similar results were obtained when probes containing regions + 9/+ 49 (single Sp1/Sp3 binding site) and + 46/+ 90 (two Sp1/Sp3 binding sites) [9] were employed (Supplementary material, Fig. 2). Furthermore, the binding of rhSp1 to the + 9/+ 49 and the + 46/+ 90 regions was decreased by phosphorylation with purified CK2 (Fig. 4B).