Fig. 4: SDF-1 inhibits RelA binding to CXCR4 promoter in vitro and in vivo.

A Nuclear protein extracts were prepared from untreated EPCs and analyzed by EMSA with the CXCR4-biotin probe. Specific competition was performed with the 50-, 100- or 200-fold molar excess of unlabeled CXCR4 probe. B EPCs were pre-treated with FGF23 for 30 min followed by SDF-1 stimulation for 10 min. Treatment of NF-κB inhibitor (Helenalin) for 40 min was used as a positive control. Nuclear protein extracts were prepared and analyzed by EMSA with the CXCR4-biotin probe. C For supershift assay, RelA, p-RelA, or p50 Antibodies were incubated with untreated EPC nuclear extracts prior to the addition of the CXCR4-biotin probe. D For DAPA assay, untreated EPC nuclear extracts were incubated with the CXCR4-biotin probe and streptavidin beads. The beads were further analyzed by western blotting with RelA, p-RelA, or p50 Antibodies. E–G EPCs were pre-treated with FGF23 for 30 min followed by SDF-1 stimulation for 15 min. Incubation of NF-κB inhibitor (Helenalin) for 40 min was used as a positive control. For ChIP assays, cross-linked DNA were immunoprecipitated with RelA (E), p50 (F), or p-RelA (G) antibodies and subjected to PCR with CXCR4 specific primers. Treatment with IgG antibody was used as a negative control. PCR product of unprecipitated DNA was used as an input control.