Fig. 1. Preparation of dsRNA and specific protein depletion.

(A) PCR fragment of ~700 bp synthesized from MAST/Orbit cDNA. (B) Corresponding dsRNA obtained by in vitro transcription using the previous PCR fragment as template. The lower band corresponds to ssRNA that did not form duplexes. (C) Monitoring of protein levels upon addition of 30 µg of dsRNA to the cells by immunoblot using MAST polyclonal antibodies and anti-α-tubulin monoclonal antibodies as a loading control. (D) Determination of the amount of dsRNA necessary to deplete MAST from S2 and Dmel2 cells. Protein levels were monitored 120 h after addition of dsRNA. It is clear that for the case of MAST, protein depletion is significantly more effective in S2 cells. (E) Addition of dsRNA to S2 cells did not cause a significant effect upon cell viability throughout the experiment.