MLL stabilizes AML1. (A) Stabilization of AML1 in 293T cells by coexpression of MLL or presence of MG132, a proteasome inhibitor. (B) Ratio of AML1/β-Actin is determined by densitometry based on the Western blot in Figure 4A. (C) Real-time PCR measurement of AML1 mRNA levels in experiment shown in Figure 4A. (D) Inhibition of AML1 poly-ubiquitination by coexpression of CBFβ and wild-type MLL but not MLL mutants. Lane 1, transfection control; lane 2, AML1-His6 with pCXN2 vector; lane 3, AML1-His6 with Flag-MLL; lane 4, AML1-His6 with Flag-MLL (Y3858N); lane 5, AML1-His6 with Flag-MLLΔSET; lane 6, AML1-His6 with CBFβ. Western blots were performed using the indicated antibodies (row 1, anti-AML1 and anti-CBFβ; row 2, anti-actin; row 3, IP with anti-Flag and IB with anti-Flag; row 4, His-tag AML1 were affinity purified with Ni-NTA magnetic Agarose beads and IB with anti-ubiquitin). (E) Diagram of a series of MLL deletion mutant constructs: MLL (1-3969), MLL (Y3858N), MLL-NC (1-3969), MLLΔSET-NC (1-3811), MLL ΔSET (1-3811), MLLn (1-2666), and MLLc (2720-3969). The arrows indicate 2 taspase I processing sites and the stars indicate the sites of the taspase I processing site mutations (cleavage site 1 mutation, D2666A/G2667A; cleavage site 2 mutation, D2718A/G2719A). (F) Interaction between AML1 and MLL deletion proteins. Lane 1, transfection control; lane 2, AML1 with pCXN2 vector; lane 3, AML1 with Flag-MLL-NC (noncleavable); lane 4, AML1 with Flag-MLL; lane 5, AML1 with Flag-MLLΔSET-NC; lane 6, AML1 with Flag-MLL-ΔSET; lane 7, AML1 with Flag-MLLn; and lane 8, AML1 with Flag-MLLc. Western blots were done using the indicated antibodies (row 1, anti-AML1; row 2, anti-actin; row 3, IP with anti-Flag and IB with anti-AML1; row 4, IP with anti-Flag and IB with anti-MLLn; row 5, IP with anti-Flag and IB with anti-MLLc). (G) Stabilization of AML1 family proteins (AML1, AML2, and AML3) by MLL. AML2 was expressed in lane 2 and 3. AML1 was expressed in lane 4 and 5. AML3 was expressed in lane 6 and 7. Flag-MLL was expressed in lane 3, 5, and 7. Western blots were done using indicated antibodies (row 1, anti-RUNX; row 2, anti-Flag; row 3, anti-actin). Spaces have been inserted in the top panel to indicate a repositioned gel lane. (H) AML2, AML1, and AML3 mRNA expression level ratios measured by real-time PCR assay in experiment of 4G. Lane 1, AML2 mRNA levels ratio between AML2 without MLL/AML2 with MLL; lane 2, AML1 mRNA levels ratio between AML1 without MLL/AML1 with MLL; lane 3, AML2 mRNA levels ratio between AML3 without MLL/AML3 with MLL.