Sox6 might repress the ɛy promoter, either through direct physical contact with the promoter or by regulating intermediates affecting the ɛy promoter. To investigate whether Sox6 is directly associated with the ɛy promoter, we first performed electrophoretic mobility shift assays (EMSA) using a c-Myc-tagged Sox6 in a reticulocyte lysate-based transcription/translation in vitro system. The probes used are listed in Figure 3A. The 36–base pair (bp) WT probe corresponds to the critical region of the ɛy promoter defined in our promoter deletion analyses. This probe contains two consensus Sox/Sox6 binding sites. Also included in our EMSA are three mutated probes that are, either truncated (M1), or mutated (M2 and M3) in Sox/Sox6 binding sites (Figure 3A). Sox6 is able to physically associate with the 36-bp region (Figure 3B) within the ɛy promoter defined by the deletion analysis experiments (Figure 2C). The 36-bp probe was shifted by the tagged Sox6 protein. Moreover, both c-Myc and Sox6 antibodies supershift the band, indicating that the binding is Sox6-specific. To rule out the possibility that the c-Myc tag itself binds to the probe, an HA-tagged Sox6 was used in another EMSA that confirmed these results (Figure 3C).