Figure 3.

dSBP2 has distinct affinities for form 1 and 2 SECIS RNAs. (A) Gel shift assays were performed between in vitro transcribed ³²P-labeled PHGPx (form 2), GPx1 and SelN (form 1) SECIS RNAs and either purified dSBP2 or hSBP2, with the range of protein concentration (nM) indicated above each gel. Proteins were omitted in lanes 1, 8, 15, 22, 30 and 38. The asterisk in lanes 8–14 is as in Figure 2A. (B) In vitro translation assay (reticulocyte lysate) of (³⁵S)-Met-labeled rat glutathione peroxidase 1 (GPx1) from reporter constructs carrying the GPx1 ORF without SECIS RNA (ΔSECIS) or with either the PHGPx, GPx1 or SelN SECIS RNAs in the 3′UTR. Purified dSBP2 or hSBP2 were omitted (lanes 1, 6, 11 and 16) or added at the indicated concentrations (nM). Translation products were treated as in Figure 2B.