P rela

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 extracts were prepared using Nuclear/Cytosol Fractionation Kit (BioVision). EMSA was performed using the LightShift Chemiluminescent EMSA Kit (Thermo Scientific) according to the manufacturer’s instructions. The double-stranded oligonucleotide containing the NF-κB consensus sequence: 5′-CAGGGTCCCCTGGGCTTCCCAAGCCGCGCACCTCT-3′ presented in the promoter of CXCR4 at −130 to −164. Oligonucleotide probe was labeled with biotin at the both 5′ and 3′-end and incubated with nuclear extracts for 20 min at room temperature. For competition experiments, unlabeled CXCR4 probe was added at a 50-, 100- or 200-fold molar excess before the labeled probe. For the supershift assay, 2 µg of RelA (Santa Cruz, sc-8008X), p-RelA (Santa Cruz, sc-136548X) or p50 (Santa Cruz, sc-8414X) antibodies were pre-mixed with the nuclear extracts for 30 min on ice before adding the binding reaction mixture, further incubated for 20 min at room temperature. The resulting DNA–protein complexes were separated from the free oligonucleotides on 6% native polyacrylamide gel and transferred onto Immobilon-Ny+ Membrane (Millipore). The membrane was incubated with Streptavidin-HRP Conjugate, and then detected by chemiluminescence.

ChIP assay was performed by SimpleChIP® Plus Enzymatic Chromatin IP Kit (Cell Signaling) according to the manufacturer’s instructions. EPCs were pre-treated with FGF23 for 30 min followed by SDF-1 stimulation for 15 min. Treatment of NF-κB inhibitor (Helenalin) for 40 min was used as a positive control. The cells were fixed with 1% formaldehyde, sonicated, and sheared into 150–900 bp of DNA fragments by micrococcal nuclease for 20 min at 37 °C. A portion of the cross-linked protein–DNA complexes were reserved as input DNA, and then the rest protein–DNA complexes were immunoprecipitated with RelA (Cell Signaling, #8242, 1:500), p-RelA (Santa Cruz, sc-136548X, 1:500), p50 (Santa Cruz, sc-8414X, 1:500), GLP (Abcam, ab41969, 1:250), H3K9me2 (Abcam, ab1220, 1:250), p300 (Abcam, ab14984, 1:250) or control (Cell Signaling, #2729, 1:500) antibodies. PCR analyses were carried out for 36 cycles with the following primer pairs: (5′-CGGACTCACTACCGACCAC-3′ & 5′-CGTCACTTTGCTACCTGCTG-3′) were used for CXCR4; (5′-GGTACATCCTCGACGGCATCT-3′ & 5′-GTGCCTCTTTGCTGCTTTCAC-3′) were used for IL-6; (5′-ACTGAGAGTGATTGAGAGTGGAC-3′ & 5′-AACCCTCTGCACCCAGTTTTC-3′) were used for IL-8; and (5′-CTACCTCCACCATGCCAAGT-3′ & 5′-GCAGTAGCTGCGCTGATAGA-3′) are used for VEGF-A.