Blocking ubiquitin-proteasome pathway does not completely rescue MLL-BP and MLL fusion–mediated RUNX1 downregulation. (A) RUNX1 expression vector was cotransfected with blank vector, full-length MLL, MLL-BP, or MLL-ENL expression vector in 293T cells. At 36 hours’ posttransfection, protein synthesis inhibitor cycloheximide (CHX) was added to a final concentration of 10 μg/mL. Cells were then harvested at indicated time points (0, 3, and 6 hours) and RUNX1 levels were determined by western blotting. β-Actin was used as a loading control. (B) Graph showing relative RUNX1 band densities normalized by β-Actin in panel A. The ratio was set to 1 at time point zero. (C) Immunoblot of transient expression of RUNX1 and CBFβ with blank vector, full-length MLL, MLL-BP, MLL-AF9, MLL-AF4, or MLL-ENL in 293T cells. Cells were treated with dimethylsulfoxide (DMSO) or 10 μmol/L MG132 for 6 hours before harvest. (D) MV4-11 and SKM1 cells were treated with DMSO (solvent, as control), 10 μmol/L MG132, or 10 μmol/L lactacystin for 6 hours. The endogenous RUNX1 and CBFβ proteins were measured by western blotting. β-Actin was used as a loading control. (E) Ubiquitination assay of RUNX1 coexpressed with MLL, MLL-BP, MLL-ENL, and MLL-AF9. (F) Schematic representation of RUNX1 (9K/R) construct used in this study. The positions of 9 lysines in RUNX1 are indicated by asterisks with numbered markers. (G) Immunoblot of wild-type (WT) RUNX1 or RUNX1 (9K/R) in which all lysines were mutated to arginines transiently coexpressed with blank vector, full-length MLL, MLL-BP, MLL-AF9, and MLL-ENL in 293T cells. (H) Immunoblot of RUNX1 in 293T cells transiently expressing WT RUNX1 or RUNX1 (9K/R) treated with DMSO or 10 μmol/L MG132 for 6 hours. All of these experiments were repeated 3 times and representative data are shown.