Figure 3. A constitutively active ubiquitin-proteasome pathway degrades Drosha.

Whole cell lysates of the indicated cell types were prepared with IP Lysis Buffer (Pierce); 20 µg protein aliquots were used for western blots. All experiments were repeated three times with similar results (p<0.05 by Student’s t-test). (A) Abundant polyubiquitination was observed in multiple cell types using lysine 48-linkage specific polyubiquitin antibody. (B) Drosha is ubiquitinated in various cell types including HEK293T, HeLa and AGS. Left panel: HEK293T lysates were immunoprecipitated with control IgG or Rabbit Polyclonal Antibody to Drosha respectively. Right panel: Cell lysates were immunoprecipitated respectively with Rabbit Polyclonal Antibody to Drosha. The immunoprecipitates were resolved by SDS-PAGE and blotted with lysine 48-linkage specific polyubiquitin antibody. The same membrane was reblotted with Drosha Rabbit mAb. (C) Inhibition of the ubiquitin-proteasome pathway with MG132 (10 µM) increases endogenous Drosha protein level in AGS cells. GAPDH was used as a loading control. (D) Proteasomal inhibition increases exogenous GFP-Drosha expression level. HEK293T cells were transfected with GFP-Drosha. Twenty-four hours post-transfection, the cells were treated with 1 µM Clasto-Lactacystin-β-lactone (Omuralide) overnight. GFP-Drosha protein level was increased 3-fold following treatment.