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. 2003 Aug 13;23(19):7415–7425. doi: 10.1523/JNEUROSCI.23-19-07415.2003

Figure 2.

Figure 2.

Specific binding of Freud-1 to 5-HT1A-FRE requires an intact CalB motif. The sequences of oligonucleotides for DRE (31 bp), FRE (14 bp), and TRE (12 bp) are indicated. A, Recombinant Freud-1-DRE binding was assessed by EMSA using 5-HT1A-DRE (31 bp) as a probe incubated with 4 μg of purified Freud-1 (lanes 1-6) or without (lane 7) as indicated. A single specific species (band 1) was retarded in the presence of 10 μg of anti-Freud-1 antibody (band 2, lane 4). The retarded complex was blocked by coincubation with 4 μg of peptide antigen (lane 6). Double-stranded unlabeled competitor oligonucleotides (at 100-fold molar excess) were included as indicated. FRE was sufficient to compete for Freud-1-DRE complex. B, Recombinant Freud-1-FRE binding: 4 μg of purified Freud-1 interacted specifically with FRE (band 1, lane 1). Freud-1-FRE interaction was abolished by preincubation with 100-fold unlabeled FRE (lane 2) but not by TRE (lane 3). C, Recombinant mutated Freud-1-DRE binding: EMSA using DRE as a probe incubated with 8 μg of purified Freud-1 with a disrupted CalB domain (Freud-1 del) alone (lane 1) or with unlabeled 14 bp FRE (lane 2) as indicated. A disrupted CalB motif reduced affinity of Freud-1 for the DRE. D, Presence of Freud-1 in nuclear extracts of L6 and RN46A cells. EMSA using the 31 bp DRE as a probe incubated without (lane 1) or with nuclear extracts from L6 (lanes 2, 3) or RN46A cells (lanes 4, 5) as indicated. The higher mobility complex (band 1) was displaced rather than retarded in the presence of anti-Freud-1 antibody in extracts from both cell lines (lanes 3 and 5, respectively). The second protein/DRE complex present in L6 cells (band 2) was not affected by Freud-1 antibody (lane 3) and represents an additional unknown repressor.