Figure 8

Syncrip selectively co-precipitates mRNAs of Cdc42, N-WASP, Arp2, Arp3 but not GAPDH and CT45. L428 cells were transfected with scrRNA or CT45 siRNA, respectively. 72 h after transfection, cell lysates were prepared and syncrip immunoprecipitations performed as detailed in Material and Methods. Protein A beads used for preclearing were used as internal control (co) for the RT-PCR or Western blot. In addition, respective RT-PCRs were also performed using unseparated cell lysates as starting material. The whole cell lysates were also used to document the mild reduction of syncrip protein by Western blotting (WB, upper panel for syncrip, short exposure; lower panel, long exposure). The RT-PCR revealed that mRNAs for Cdc42, N-WASP, Arp2 and Arp3 (but not CT45 or GAPDH) could be amplified from syncrip-immunoprecpitations. In addition, the amount of co-precipitated mRNA was reduced in CT45 siRNA-treated cells.