Figure 5
EMSA assays showing direct binding of NRF1 and ZSCAN10 to the SIX1 promoter in vitro. (a) Sequence and putative transcription factor-binding sites in the wild type (WT) and mutation type (MT) in the proximal minimal promoter of SIX1 gene. The putative transcription factor binding sites are boxed. The primers for the unidirectional deletions are underlined. (b) Nuclear protein extracts were incubated with a free probe containing the NRF1 binding site in the presence or absence of any competition (lane 2), 50× mutation probe (lane 3) and 50× unlabelled probe (lane 4). The super-shift assay was conducted using 10 μg anti-NRF1 antibodies (lane 5). (c) Nuclear protein extracts were incubated with a free probe containing the ZSCAN10 binding site in the presence or absence of any competition (lane 2), 25× mutation probe (lane 3) and 25× unlabelled probe (lane 4). The super-shift assay was conducted using 10 μg anti-ZSCAN10 antibodies (lane 5). The arrows mark the main complexes.
