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Figure 4


Fig. 4. Deletion of the putative antizyme-binding site and effect on antizyme binding. (A) Schematic of wild-type AZI and mutant AZI{Delta}117-140. The putative antizyme(AZ)-binding site, designated residues 117-140, was deleted using overlap extension PCR. (B) Western blot analysis of wild-type AZI and AZI{Delta}117-140 using AZI antibody. Proteins were synthesized using in vitro transcription and translation from pTriEx-Hygro-AZI and TriEx-Hygro-AZI{Delta}117-140 constructs as S-tagged fusion proteins. (C) Loss of antizyme-binding by AZI{Delta}117-140. The ability of S-tagged AZI or AZI{Delta}117-140 to associate with radiolabeled antizyme was examined by affinity purification using S-protein agarose beads, followed by SDS-PAGE analysis and autoradiography. The mutant AZI{Delta}117-140 was unable to bind antizyme.





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