Figure 5

(a) EMSA detection of direct binding of purified GST-PAX3-FKHR fusion protein (left panel) and nuclear extract from PAX3-FKHR-expressing C2 cells (right panel) to wild-type (nWT) and mutant (nM1 and nM2) DNA probes. Arrow indicates migration of PAX3-FKHR-DNA complexes. Presence of PAX3-FKHR in the bound protein complexes was detected by antibody super-shift. (b) Chromatin immunoprecipitation analysis detected specific in vivo interaction between PAX3-FKHR and CCN3 gene. Chromatin was prepared from C2 cells expressing PAX3-FKHR (left and middle panels) or Pax3 (right panel) as described in Materials and methods, and subjected to IP using an antibody that recognizes both Pax3 and PAX3-FKHR. Histone H3 and normal rabbit IgG antibodies (provided by SimpleChIP enzymatic chromatin IP kit) were used in separate reactions as positive (RPL3, ribosomal protein L3) and negative controls for the chromatin immunoprecipitation assay, respectively. Input represented 10% of the chromatin preparation before IP. Opposing arrows indicate the locations of the CCN3 promoter primer sets. Expected sizes for RPL3 and CCN3 PCR products were 161 and 191 bp, respectively.