Figure 2.

MLL undergoes bimodal degradation by the UPS. (A) The levels of MLL mRNA do not correlate with the biphasic MLL protein expression through specific phases of the cell cycle. qRT–PCR was performed on HeLa cells that were synchronized by double thymidine treatment and released for the indicated periods of time. MLL expression was normalized against GAPDH. The average MLL expression in asynchronous HeLa cells was arbitrarily assigned as 1.0. Values shown are mean ± 1 SD obtained from three independent experiments. (B) MLL is degraded at specific phases of the cell cycle. On-bead in vitro degradation assays were carried out by incubating purified Flag-MLL^N320/C180 with cellular lysates prepared from cells synchronized at the indicated phases of the cell cycle for 30 min at 30°C. The abundance of Flag-MLL was analyzed by anti-Flag immunoblots. (C) The degradation of MLL can be prevented by inhibitors of the 26S proteasome. HeLa cells were treated with 10 μg/mL cycloheximide for the indicated periods of time in the presence of either DMSO as a control or 10 μM MG132 to inactivate 26S proteasome. (Left panel) The expression of MLL was evaluated by anti-MLL immunoblots. The expression of β-actin served as a loading control. (Right panel) The half-life of MLL^N320/C180 was determined by quantifying the levels of normalized MLL^C180. (D) MLL undergoes polyubiquitination. 293T cells transfected with indicated expression vectors for 48 h were treated with MG132 or DMSO vehicle for an additional 24 h. Cells were lysed in RIPA buffer containing 20 mM N-ethylmaleimide, which inhibits deubiquitination. Cellular extracts were subjected to anti-Flag immunoprecipitation, SDS-PAGE, and immunoblots with the indicated antibodies.