Figure 3.
Silencing CUL4, DDB1, or Fbw5 stabilizes TSC2. (A) Silencing DDB1, FBW5, or a combination of CUL4A and CUL4B in mammalian cells increases the half-life of endogenous TSC2 protein. U2OS cells transfected with siRNAs targeting the indicated genes were treated with cycloheximide for different times. The steady-state level of endogenous TSC2 as well as TSC1 was determined by direct immunoblotting. (B) Silencing DDB1, FBW5, or a combination of CUL4A and CUL4B in mammalian cells increases the half-life of ectopic TSC2 protein. U2OS cells were transfected with siRNAs targeting the indicated genes 12 h after transfection of plasmid expressing HA-TSC2. The half-life of HA-STC2 was measured by 35S pulse-chase assay.