Fig. 6.
Binding of transcription factor NF-1 to the mGITRL promoter. (A) The presence of cis-acting elements between -120 and -94 is suggested by luciferase assays (Fig. 5B). Oligo probes P1 to P3 for EMSA were designed in this identified region and its flanking regions. The locations of these probes are indicated in Fig. 5A. EMSA was performed by using the probes (P1-P3) and nuclear extract from RAW 264 cells. (B) A competition assay was performed by using a 100-fold excess of unlabeled competitor with 32P-labeled P2 probe. The competitors used are indicated above the gel. Probe P2 sequence and mutated sequences in M1 and M2 are shown under the gel. Sequence similar to NF-1 consensus in P2 is indicated with underlines. (C) Super shift assay was performed by using probe P2 and an anti-NF-1 antibody (marked +). (D) EMSA was performed by using probe P2 and nuclear extracts from nonstimulated (0 h) and LPS-stimulated (2-24 h) RAW 264 cells. NF-1 and probe complexes are indicated. (E) EMSA was performed by using probe P2 and nuclear extracts from nonstimulated (0 h) and LPS-stimulated (2-24 h) bmDC (EMSA). NF-1 in nuclear extracts used for EMSA was detected by immunoblotting using anti-NF-1 antibody (IB).