Fig. 4.

Effects of the transposon-derived Ex4 coding region in in vitro DNA binding and transrepression activities of DM-W. (A) In vitro DNA binding of DM-W (full length) and its C-terminal truncated protein, DM-W (Δ124–194), to the DMRT1-binding sequence. Flag-DM-W(full) and Flag-DM-W(Δ124–194) were produced by in vitro transcription–translation system and analyzed by Western blot analysis with an anti-FLAG antibody followed by an HRP-conjugated antimouse antibody (left). The relative intensity values were shown below. EMSA was performed using in vitro synthesized Flag-DM-W(full) (0.5, 1.0, 2.0, or 3.0 μl) or Flag-DM-W(Δ124–194) (2.0, 4.0, or 5.5 μl) and ³²P-labeled double-stranded oligonucleotides containing the DMRT1-binding sequence. The relative intensity values to the protein amounts, binding strength, and ratio of strength/protein amount were shown below. (B) The luciferase reporter assay for the DM-W transrepression activity on DMRT1-driven transcription. The DMRT1-driven luciferase reporter and an expression plasmid for DMRT1.S and DM-W(full) or DM-W(Δ124–194) were transfected into HEK293T cells and posttransfection (24 h) luciferase activity was measured. Letters (a–d) indicate significant differences based on a one-way ANOVA, followed by the Tukey–Kramer HSD test (P < 0.01).