Journal:

Article Title: The Zinc Finger Antiviral Protein Directly Binds to Specific Viral mRNAs through the CCCH Zinc Finger Motifs

doi: 10.1128/JVI.78.23.12781-12787.2004

Figure Lengend Snippet: In vitro binding of ZAP to target RNA. The lysates of ZAP-expressing or control cells were mixed with 9E10 anti-Myc antibody and protein G-agarose resin for 2 h to immobilize ZAP to the resin. The resins were washed and incubated with the indicated 32P-labeled RNA probes for 30 min in 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 with the 9E10 antibody. (A) Binding of RNAs to NZAP-Zeo-myc. The NZAP-Zeo-myc protein was expressed in Rat2 cells and analyzed for binding to the indicated RNA probes. Rat2-vector, lysate of Rat2 cells transfected with the empty vector pcDNA4/TO-myc-His; Rat2-NZAP-Zeo-myc, lysate of Rat2 cells transfected with pcDNA4/TO/myc-NZAP-Zeo. The arrow in the lower panel indicates the position of NZAP-Zeo-myc. (B) Binding of RNAs to full-length ZAP. 293TRex, lysate of 293TRex control cells treated with tetracycline; 293TRex-ZAP, lysate of 293TRex-ZAP cells treated with tetracycline. The arrow in the lower panel indicates the position of ZAP-myc. (C) A mutation in the second zinc finger of ZAP abolished protein binding to the target RNA. 293TRex-ZAP-C88R, lysate of 293TRex cells expressing the second zinc finger mutant ZAP-C88R. The arrow in the lower panel indicates the position of ZAP-C88R-myc.

Article Snippet: The Myc-tagged proteins were immobilized to protein G-agarose resin (Santa Cruz) by use of the 9E10 antibody (Santa Cruz).

Techniques: In Vitro, Binding Assay, Expressing, Incubation, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Western Blot, Plasmid Preparation, Transfection, Mutagenesis, Protein Binding