Mutations in the DBD impair DNA binding. NR2E3 wild-type and mutants proteins were in vitro transcribed/translated in reticulocyte lysates (TNT; Promega).; In vitro DNA-binding of NR2E3 proteins was tested on a radiolabeled consensus DR1 response element [1] with a 100-fold excess of either non-specific (−) or specific (+) competitor oligonucleotides. One μl of programmed reticulocyte lysate was used per binding reaction. Transcription/translation efficiency was tested by western blot analysis (data not shown). The migration of the complex formed by binding of the NR2E3 dimer to the probe is indicated by a black arrow. DNA binding is only detected in the presence of wild-type NR2E3 and the LBD-mutant p.R311Q (low level of detection was observed for the p.R311Q mutant in this experiment, but higher exposition and published studies [21] showed clearly a DNA-binding for the p.R311Q mutant). All DBD mutants, i.e. p.G56R, p.R76Q, p.R76W, p.G88V, p.R97H, p.R104Q and p.R104W, did not bind to DNA. The radiolabeled probe was obtained by annealing oligonucleotides NR2E3REfor (5′-CCTTTAAAAGTCAAAAGTCAACTTCCAA-3′) and NR2E3RErev (5′-TTCCGTTGGAAGTTGACTTTTGACTTTT-3′). Radiolabeling by Klenow fill-in with 30 μCi of [α-32P]dATP (3000 Ci/mmol) (Hartmann Analytik, Braunschweig) and subsequent probe purification on Sephadex G-50 columns was according to manufacturers instructions (Roche, Basel, Switzerland). DNA-binding reactions were carried out in 20 μl of 10 mM Tris (pH 7.5), 160 mM KCl, 1 mM dithiothreitol (DTT), 10% glycerol, 10 μg of sonicated salmon sperm DNA (Roche), 2 μg of poly(dI-dC) and 1 μl of programmed reticulocyte lysate. After a 15-min incubation on ice, 1 ng of 32P-labeled probe was added, and incubations were continued for an additional 15 min at room temperature. DNA-protein complexes were separated from free probe on a native 4% polyacrylamide gel in 0.5 x Tris/borate/EDTA (TBE) buffer. Gels were dried and revealed by phosphorimaging (GE Healthcare, Piscataway, NJ).
http://plosone.org/article/info:doi/10.1371/journal.pone.0007379#article1.body1.sec5.supplementary-material6.caption1.p1

Some of the symbols in legend s6 do not appear. The correct legend should read: Mutations in the DBD impair DNA binding. NR2E3 wild-type and mutants proteins were in vitro transcribed/translated in reticulocyte lysates (TNT; Promega).; In vitro DNA-binding of NR2E3 proteins was tested on a radiolabeled consensus DR1 response element [1] with a 100-fold excess of either non-specific (-) or specific (+) competitor oligonucleotides. One μl of programmed reticulocyte lysate was used per binding reaction. Transcription/translation efficiency was tested by western blot analysis (data not shown). The migration of the complex formed by binding of the NR2E3 dimer to the probe is indicated by a black arrow. DNA binding is only detected in the presence of wild-type NR2E3 and the LBD-mutant p.R311Q (low level of detection was observed for the p.R311Q mutant in this experiment, but higher exposition and published studies [21] showed clearly a DNA-binding for the p.R311Q mutant). All DBD mutants, i.e. p.G56R, p.R76Q, p.R76W, p.G88V, p.R97H, p.R104Q and p.R104W, did not bind to DNA. The radiolabeled probe was obtained by annealing oligonucleotides NR2E3REfor (5'-CCTTTAAAAGTCAAAAGTCAACTTCCAA-3') and NR2E3RErev (5'-TTCCGTTGGAAGTTGACTTTTGACTTTT-3'). Radiolabeling by Klenow fill-in with 30 μCi of [α-32P]dATP (3000 Ci/mmol) (Hartmann Analytik, Braunschweig) and subsequent probe purification on Sephadex G-50 columns was according to manufacturers instructions (Roche, Basel, Switzerland). DNA-binding reactions were carried out in 20 μl of 10 mM Tris (pH 7.5), 160 mM KCl, 1 mM dithiothreitol (DTT), 10% glycerol, 10 μg of sonicated salmon sperm DNA (Roche), 2 μg of poly(dI-dC) and 1 μl of programmed reticulocyte lysate. After a 15-min incubation on ice, 1 ng of 32P-labeled probe was added, and incubations were continued for an additional 15 min at room temperature. DNA-protein complexes were separated from free probe on a native 4% polyacrylamide gel in 0.5 x Tris/borate/EDTA (TBE) buffer. Gels were dried and revealed by phosphorimaging (GE Healthcare, Piscataway, NJ).