In vitro interaction domain mapping of pVHL and AUF1. In vitro interaction domain mapping of pVHL and AUF1. A, each AUF1 isoform (as indicated) was translated in vitro in the presence of 35S-methionine and tested for binding to GST or GST-VHL proteins that had been prebound to glutathione-Sepharose. GST, binding to GST alone; GST-VHL, binding to the fusion protein. B, schematic diagram of pVHL indicating the portions of the protein forming the α and β domains and the first 4 of the β sheets (arrows 1–4). p45AUF1 was translated in vitro in the presence of 35S-methionine and tested for binding to GST or GST-VHL (full-length or progressive C-terminal pVHL deletions). The amino acid at the pVHL C-terminal deletion endpoint is indicated above each lane. Inputs represent 10% of the amount used in each binding assay. C, schematic diagram of p45AUF1 indicating 2 RNA recognition motifs (RRM1, RRM2) and a glutamine-rich sequence [Q, (QYQQQQQ)]. The cross-hatched areas represent sequences encoded by the alternatively spliced exons 2 and 7. GST-AUF1 constructs used in these studies are indicated, with the amino acid numbering system corresponding to p45AUF1. Full-length p25VHL was translated in vitro in the presence of 35S-methionine and tested for binding to GST or the indicated GST-AUF1 constructs that were prebound to glutathione-Sepharose beads. D, the indicated pVHL deletion mutations were translated in vitro in the presence of 35S-methionine and tested for binding to GST-AUF1 construct # 3. 1–213, full-length pVHL; 54–213, deletion of amino acids 1–53; Δ114–155, deletion of amino acids 114–155; Δ60–114, deletion of amino acids 60–114. Inputs represent 10% of the amount used in each binding assay. E, schematic diagram of p45AUF1 indicating pVHL binding domains. Hong Xin et al. Mol Cancer Res 2012;10:108-120 ©2012 by American Association for Cancer Research