FIG. 6.

FUS binds with MnSOD promoter. The binding of FUS to the promoter of MnSOD gene was evaluated by chromatin immunoprecipitation (ChIP) assay as described under “Experimental Procedures.” Briefly, chromatins were immunoprecipitated by FUS antibody or immunoglobulin (IgG) as control and immunoprecipitated DNA was amplified by PCR using primers targeted to the promoter region (−2931 to −2712 and −2264 to −2075) of MnSOD gene. Total genomic DNA obtained after transfection was parallel-amplified by using the same primer as input control (A). Predicted putative FUS binding element in MnSOD promoter and three mutated oligonucleotides were designed to use as probe (B). Oligonucleotides were radioactively labeled with ^[32P]ATP and T4 polynucleotide kinase as described previously (23). Electrophoretic mobility shift assay (EMSA) was carried out to separate DNA-protein complexes on 6% polyacrylamide native gel. The arrow points to protein-DNA complexes (C). The biotinylated oligonucleotides of wild-type and mutant type probes were used for DNA protein interaction by affinity chromatography followed by Western blotting using anti-FUS or anti-Flag antibody (D).