ASB2 leads to reduced MLL protein level and transactivation ability. (A) MLL was coexpressed with Flag tagged ASB2, 6 and 7 in 293 cells. Western blotting with whole cell lysate shows that ASB2 specifically degrades MLL. β-actin blot shows equal loading of the samples. (B) Expression of ASB2 in 293 cells followed by Western blotting with whole cell lysate shows that ASB2 leads to degradation of endogenous MLL. (C) Equimolar amounts of MLL or MLL-AF9 expression plasmids were cotransfected with ASB2 at the ratio of 6:1, 2.5, and 4 in 293 cells. Western blotting with whole cell lysate shows that MLL degradation is ASB2 dose-dependent, wherease the levels of MLL-AF9 are not affected. β-actin blot indicates equal loadings. (D) Schematic diagram of the Hoxa9-LUC reporter. (E) Dual luciferase assay was performed in 293 cells with Hoxa9-LUC reporter. Lanes 3 through 6 show expression of MLL with increasing dosage of ASB2, and lane 7 through 10 show expression of MLL-AF9 with increasing dosage of ASB2. The ratios between MLL or MLL-AF9 and ASB2 were the same as in panel C. All changes are normalized to lane 1, which includes Hoxa9-LUC and an empty expression vector. Error bars indicate SD. Results of 1 of more than 3 representative experiments performed are shown. (F) ChIP assay was performed in 293 cells transfected with Hoxa9-LUC, MLL, and ASB2. The ratio between MLL and ASB2 was 6:4. Probes 4 through 8 cover the promoter region of Hoxa9-LUC. Probe3 recognizes a region that exists in the endogenous Hoxa9 promoter but is not included in Hoxa9-LUC, and serves as a negative control. The nomenclature is consistent with Figure 6E, which shows the position of the probes on endogenous Hoxa9 locus. Error bars indicate SD. Results of 1 of more than 3 representative experiments performed are shown.