Western blotting was performed, as previously described (18 (link), 19 (link)). Briefly, cells were lysed with RIPA lysis buffer and centrifuged for 15 minutes at 4°C. Protein concentration was assessed using a Bio-Rad protein assay kit. The protein was loaded into gels (20 μg/well), and the bands were separated using electrophoresis. Bands were transferred to nitrocellulose membranes, blocked with 5% milk for 1 hour at ambient temperature, and incubated with primary antibodies against PACT or Dicer (1:1000 dilution) overnight at 4°C. Samples of proteins were incubated with horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies (GE Healthcare) or an anti-goat antibody (Santa Cruz Biotechnology) for 1 hour at ambient temperature. Blots were developed and analyzed, as described previously (18 (link), 19 (link)). Loading control (actin) was used; all experiments were performed in duplicate.