Figure 3. Association of DJ-1 with RREB1 on the LDLR promoter.

A. Chromatin immunoprecipitation assays were carried out using chromatin prepared from SH-SY5Y cells. Chromatin was immunoprecipitated with anti-DJ-1, anti-RREB1 and anti-Sp1 antibodies or non-specific IgG. After extraction of DNA from precipitated chromatin, two regions spanning −1579 to −1291 and spanning −302 to −14 were amplified by PCR with specific primers and with amplified DNA and were separated on agarose gels as described in Materials and methods. n = 3. B. Proteins in SH-SY5Y nuclear extracts were immunoprecipitated with anti-RREB1, anti-DJ-1 and anti-Sp1 antibody or IgG. Immunoprecipitates were then analyzed by Western blotting with anti-RREB1, anti-DJ-1 and anti-Sp1 antibodies. n = 3. C. Nucleotide sequences used for labeled probe and competitors are shown (C-a). EMSA was carried out using SH-SY5Y cell nuclear extracts, IRDye800-labeled wild-type prove and non-labeled wild-type or mutated RRE competitors as described in Materials and methods (C-b). n = 3. MSA was carried out using SH-SY5Y cell nuclear extracts and 100 ng of recombinant DJ-1 on IRDye800-labeled wild-type prove (C-c). D. SH-SY5Y cell nuclear extract was first incubated with the IRDye800-conjugated probe and then incubated with 2 µg of anti-DJ-1, anti-RREB1 and anti-Sp1antibodies or non-specific IgG for 30 min at 4°C and subjected to EMSA. n = 3. E. Schematic model of DJ-1, RREB1 and Sp1 on the CCK promoter. DJ-1 complexed with RREB1 and Sp1 bind to the RRE and Sp1 site, respectively, to activate the CCK promoter.