Physical interaction between MLL and AML1. (A) Interaction between the endogenous MLL and AML1 proteins in HEL cells. HEL cell nuclear lysates were used for immunoprecipitation (lane 1-2) with IgG and anti-AML1 polyclonal antibodies cross-linked with protein A sepharose beads followed by Western blot with the indicated antibodies. Nuclear lysates prepared from 293T cells that coexpress Flag tagged MLL and AML1 were used as an IP control (lane 3) with the anti-AML1 polyclonal antibodies cross-linked with protein A sepharose beads. (B) Interaction of MLL and AML1 when both are expressed in 293T cells in the absence or presence of CBFβ, followed by immunoprecipitation (IP) and Western blot. The transfections were indicated as in lane 1, vector alone; lane 2, Flag tagged MLL and CBFβ; lane 3, Flag tagged MLL and AML1; and lane 4, Flag tagged MLL, AML1, and CBFβ. Row 1 indicates AML1 input, row 2 indicates CBFβ input, row 3 indicates Flag tagged MLL IP and Western blot, row 4 indicates Western blot of AML1 with IP samples in row 3, and row 5 indicates Western blot of CBFβ with IP samples in row 3. (C) Diagram of a series of AML1 N-terminal deletion constructs: AML1 (1-453, 25-453, 51-453, 60-453, 90-453, and 106-453), and the strength of their interaction with MLL as indicated (either – or + derived from experiment in panel E). (D) Determination of the MLL interacting domain in AML1 by IP and Western blot using the N-terminal deletion constructs of AML1 shown in panel C. (E) Competitive interaction between MLL and full-length AML1 and coexpressed N-terminal deletion constructs of AML1 in 293T cells. The upper band in lanes 1-5, top panel indicate AML1 (1-453), while the lower band in lanes 1-5 indicate AML1 (25-453, 51-453, 60-453, 90-453, and 106-453). The bands (both upper and lower) in lanes 1-3, bottom panel indicate the same bands as the top panel, whereas lanes 4-5 indicate only the AML1 (1-453) band, but not the AML1 (90-453 or 106-453) bands.