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. 2010 Feb 26;285(18):13885–13895. doi: 10.1074/jbc.M109.081463

FIGURE 2.

FIGURE 2.

A, EMSA competition assay demonstrates that the 18-bp sequence contains the RFX1-binding site. EMSA experiments using 26 bp (−492 to −467) as a probe and 10 μg of U-1240 MG nuclear extracts revealed several complexes (lane 1). The specificity of complexes formation was confirmed by 18-bp (−484 to −467) competition. Three specific complexes, which were competed by the 18 bp but not the nonrelevant competitor, AP-1, were designated as complexes A, B, and C, respectively (lanes 3 and 4). The oligonucleotides, PyEP or MAP1A, containing the known RFX1-binding site could diminish the specific complexes A and B (lanes 6 and 7) but not the oligonucleotides, 18-bp mut (lane 5), with mutated RFX1-binding core sequence. B, EMSA supershift assay reveals the in vitro interaction between RFX1 and the 18 bp in nuclear extracts (N.E.) isolated from human U-1240 MG cells. EMSA experiment was carried out using MAP1A, which contains the RFX1-binding site and human 26-bp probes. Both complexes A and B were formed with the two probes (lanes 1 and 4), diminished by human 18-bp competition (lanes 2 and 5), and supershifted by anti-RFX1 antibody (lanes 3 and 6). The result of EMSA supershift assay directly demonstrated the in vitro interaction between 18 bp and RFX1. The bands supershifted by anti-RFX1 antibody are designated as ss1. C-i, expression analyses of FGF-1B transcript in human U-1240 MG and mouse KT98 cells. Reverse transcription-PCR experiments conducted using primer specific for mouse or human FGF-1B transcripts, respectively, demonstrated the expression of FGF-1B transcripts in U-1240 MG and KT98 cells. C-ii, EMSA supershift assay reveals the in vitro interaction between RFX1 and the m18 bp in nuclear extracts isolated from mouse KT98 cells. EMSA experiment was carried out using MAP1A, which contains the RFX1-binding site, and mouse 26-bp probes. Both complexes A and B were formed with these two probes (lanes 1 and 4), diminished by mouse 18-bp competition (lanes 2 and 5), and supershifted by anti-RFX1 antibody (lanes 3 and 6). The result of EMSA supershift assay directly demonstrated the in vitro interaction between mouse 18 bp and RFX1. The bands supershifted by anti-RFX1 antibody are designated as ss1.