Figure 5.

Full-length T220D Pit-1 generated by in vitro transcription and translation also reveals differential binding at monomeric RRE and dimeric FPI sites. The full-length WT Pit-1 and mutant proteins (in pGem4 vectors) were prepared using the TNT coupled transcription-translation reticulocyte lysate system with T7 polymerase (Promega protocol). A, Representative in vitro transcription and translation reaction of Pit-1 incorporating [³⁵S]methionine. The resulting reaction was electrophoresed on a 15% SDS-PAGE gel and visualized by autoradiography. B, In each 20-μl reaction, 2 μl of sample was incubated with 28,000 cpm of the RRE, FPI, or FPIII probe in binding buffer and 0.5 μg poly deoxyinosinic deoxycytidylic acid. To the reaction, specific competitor was added (5.4 pmol RRE, 5.6 pmol FPI). After a 30-min incubation at room temperature, 2 μl of loading buffer was added to each sample and ran on preelectrophoresed 6% nondenaturing polyacrylamide gel in 0.25× TBE buffer at 190 V. After electrophoresis, the gel was dried and visualized by autoradiography. Monomeric and dimeric binding is indicated. MW, Molecular mass; NS, nonspecific binding.