Figure 1.

RNAi knockout of PP2A, PP4, and PP5 in Drosophila S2 cells. (A) Drosophila S2 cells were incubated in the presence of dsRNA corresponding to EGFP or the phosphatase subunits indicated at the top. After 72 h, cells were lysed in RIPA buffer containing 0.2 mM Na₂VO₄ and 10 mM NaF₂, and lysates were clarified by centrifugation. Equal amounts of protein (30–60 μg) were separated on SDS-polyacrylamide gels, and detected by Western blotting with antibodies against the proteins indicated on the right. (B) Drosophila S2 cells were incubated in the absence or presence of dsRNA corresponding to EGFP or the phosphatase subunits indicated at the top. After 72 h, RT-PCR was performed as described under Materials and Methods, with primers against the phosphatase subunits indicated at the right. Individual reactions (5 μl of the final reaction mixture) were resolved on a 1% agarose gel and detected by staining with ethidium bromide. Results shown are representative of three to seven experiments.