Figure 2

The activity of the ZAP mutants to bind the target RNA. The lysates of Rat2 cells expressing the indicated NZAP-Zeo-myc mutants were mixed with 9E10 anti-Myc antibody and proteinG-agarose resin for 2 h to immobilize ZAP to the resin. The resins were washed and incubated with the indicated 32-P labeled. RNA probes for 30 minutes in binding buffer and then washed three times with the binding buffer. Bound RNAs were eluted by boiling in RNA sample buffer, subjected to urea-polyacrylamide gel electrophoresis, and detected by autoradiography. Bound ZAP proteins were eluted by boiling in protein sample buffer and detected by Western blotting. Rat2-HAZ: Rat2 cells transduced with empty vector; Rat2-NZ: Rat2 cells expressing wild-type NZAP-Zeo-myc; Nm: Rat2 cells expressing NZAP-Zeo-myc mutants; C88R: cells expressing full-length ZAP-C88R-myc mutant as a negative control.