Figure 1. Acetylation of Drosha increases its protein level.

All experiments were repeated three times with similar results (p<0.05 by Student’s t-test). (A), Inhibition of deacetylases increases Drosha protein level measured by western blot. HEK293T cells were treated with vehicle, trichostatin A (TSA, 2 µM), or nicotinamide (NIA, 1 mM) overnight prior to harvest. (B) TSA, an HDAC inhibitor, increases ectopically expressed Drosha levels in HEK293T upon transfection with a GFP-Drosha construct. (C) Inhibition of deacetylases have no effects on Drosha mRNA level measured by RT-PCR. (D)Inhibition of deacetylases increases Drosha acetylation. HEK293T cells were transfected with empty vector (EV) or GFP-Drosha. Twenty-four hours post-transfection, the cells were treated with TSA (2 µM) or NIA (1 mM) overnight. Whole cell lysates were prepared and immunoprecipitated with GFP antibody conjugated sepharose beads. The immunoprecipitates were resolved and blotted with mouse monoclonal acetylated lysine antibody to detect Drosha acetylation. The same membrane was then reblotted to check Drosha protein level. (E) Multiple acetyl transferases acetylate Drosha. A GFP-Drosha construct was cotransfected with an empty vector, p300, CBP, PCAF or GCN5 construct respectively into HEK293T cells. Forty-eight hours post-transfection, half of the cells was used to extract total RNA for checking the mRNA levels of GFP-Drosha. Another half of the cells was used to prepare whole cell lysates for detecting Drosha acetylation as in Figure1C.