Fig. 4.

EMSA supershift analysis of the IR5 with PU.1 and competition with mutant oligos. (A) Nuclear extracts from untransfected HEK 293T cells (lane 2) or cells transfected with a vector for HA-PU.1 (lanes 3–8) were incubated with radiolabelled IR5 DNA alone or in the presence of antibodies against PU.1, Ets-1, Elk-1, the HA epitope and STAT3, as indicated. PU.1 complexes are indicated with an arrow. The partial supershift with the HA antibody likely reflects a degree of translation initiation at an internal AUG. (B) A recombinant HA-tagged Ets domain of PU.1 was incubated with IR5 DNA alone (lane 2), in the presence of anti-HA antibody (lane 3) or 100-fold excess of unlabelled wt or mutant IR5 DNA (lanes 5–7). In parallel, a recombinant HA-tagged Ets domain of Elk-1 did not form a complex with IR5 DNA (lane 4).