EMSAs of PtAu1afull in the dark in the absence and presence of 10 mM MgCl2 in the polyacrylamide gels as well as in the reaction and running buffers. The 35-bp DNA probe used in the EMSAs was the one used for DNA binding studies of VfAu1 in the publication by Takahashi et. al. (Takahashi et al., 2007) (5'-GGAGTATCCAGCTCCGTAGCTGACGTG GCCTCTGG-3', the bZIP target sequence is underlined). The DNA probe (388 nM) was incubated in buffer D without MgCl2 (a) and with MgCl2 (b) with varying amounts of purified PtAu1afull (2, 4, 7.9, 15.9, 31.8, 63.5, 125, 250, 500, 1000, 2000, 4000 nM). EMSA runs were performed as described in the methods section. In the EMSA in the absence of MgCl2, the formation of a PtAu1afull-DNA complex is already observed at the lowest protein concentration of 2 nM. At protein concentrations above 250 nM, a second PtAu1afull-DNA band appears, which was also observed in gel shift experiments performed by Takahashi et. al. (Takahashi et al., 2007) and in DNA binding studies of the CREB bZIP domain (Moll et al., 2002). This slower migrating band most likely represents two PtAu1afull dimers bound to the 35-bp DNA probe, indicating the ability of unspecific DNA binding of PtAu1afull at high protein concentrations. In the EMSAs performed in the presence of MgCl2, the formation of a stable PtAu1afull-DNA complex is observed at much higher protein concentrations compared with the experiments without MgCl2. Additionally, the slower migrating PtAu1afull-DNA band disappeared, indicating sequence-specific DNA binding of a single PtAu1afull dimer to the target DNA sequence. Therefore, it can be concluded that the presence of MgCl2 is required for sequence-specific DNA binding of PtAu1afull. bZIP, basic region leucine zipper; EMSAs, electrophoretic mobility shift assays.