The RUNX1 C-terminus is required to exclude corepressors from the RUNX1/PU.1 complex. (A) Increasing concentrations of RUNT did not prevent the co-IP of ETO2 or SIN3A with PU.1. 293T cells were transfected with HA-ETO2, Flag-PU.1, and increasing amounts of HA-RUNT. Increasing amounts of RUNT did not decrease co-IP of ETO2 or endogenous SIN3A with PU.1 (lanes 5-8). The vertical line separates repositioned gel lanes (lanes 9-11) that showed anti-Flag did not IP HA-RUNT, HA-ETO2, or SIN3A in the absence of Flag-PU.1. Dashed outline shows expected position of protein in these lanes. (B) In the reverse co-IP experiment, increasing amounts of RUNT did not prevent the co-IP of PU.1 with ETO2. 293T cells were transfected with HA-ETO2, PU.1, and increasing amounts of Flag-RUNT. Increasing amounts of RUNT did not decrease the co-IP of PU.1 with ETO2 (lanes 5-8). The vertical line separates repositioned gel lanes (lanes 9 and 10), which showed that anti-HA did not immunoprecipitate PU.1 or Flag-RUNT in the absence of HA-ETO2. Dashed outline shows expected position of protein in these lanes. (C) In contrast, increasing amounts of RUNX1 decreased co-IP of PU.1 with ETO2. 293T cells were transfected with expression vectors for HA-ETO2, PU.1, and flag-RUNX1. PU.1 co-IPs with ETO2 (lane 5). Increasing amounts of RUNX1 decreased co-IP of PU.1 (lanes 6-8). The vertical line separates repositioned gel lanes (lanes 9 and 10) that showed anti-HA did not IP PU.1 or Flag-RUNX1 in the absence of HA-ETO2. Dashed outline indicates expected position of protein in these lanes.