Misguided Transcriptional Elongation Causes Mixed Lineage Leukemia (original) (raw)
Figure 3
Incorporation of MLL fusion proteins into EAP.
(A) Coimmunoprecipitation of MLL-ENL with EAP components. MLL-ENL (MLL-ENL), a MLL-ENL mutant lacking the last 15 amino acids of ENL (MLL-ENL1–544), or MLL without fusion partner (MLL) were expressed either alone or together with HA-tagged proteins AF4, AF5, or Dot1l. A schematic overview of the EAP core structure in the presence of MLL-ENL including the expected protein–protein interactions (double-headed arrows) is depicted in the upper-left panel. The presence of HA-tagged proteins and endogenous CDK9 in anti-MLL precipitates was probed alongside with a sample of the input (inp, 5%) by immunoblots as indicated. As a control, the successful precipitation of the MLL-ENL derivatives was confirmed by an anti-MLL blot. (B) Interaction of MLL-AF4 and MLL-AF5 with endogenous (endo) ENL and CDK9. MLL-AF4 (MLLAF4758–1210) and MLL-AF5 (MLLAF5731–1163) proteins analogous to patient-derived fusions were expressed in 293T cells. Note that the N-terminal CYCT interaction domain is missing in leukemogenic MLL-AF4/5 fusions as depicted in the upper-left and -right panels. MLL-AF4/5 derivatives deleting the ENL binding domain (MLLAF41023–1210, MLLAF5991–1163) served as controls. The coprecipitation of endogenous ENL and CDK9 was detected by immunoblot as indicated next to a sample of 5% input (inp). The presence of MLL-AF4/AF5 in the lysates was controlled as above.