MC3T3 cells were transfected with siRNA and/or stimulated with TNFα (10 ng/mL) for 1 hour. ChIP assays were performed using ChIP-IT kit (Active Motif, Carlsbad, CA, USA).(39 (link)) Isolated DNA was subjected to real-time PCR using specific primers from IDT.
For some of the experimental studies, the cells were washed and incubated with Wnt3a (100 ng/mL) or Bmp2 (100 ng/mL) for 4 hours. About 45 minutes before stimulation, some of the cells were incubated with TNFα (10 ng/mL). After stimulation, protein synthesis was inhibited by incubating the cells with cycloheximide for 3 hours, after which ChIP assay was carried out. The chromatin was immunoprecipitated using antibodies for p65 (Active Motif), p50 (Cell Signaling Technology), HDAC1 (Active Motif), β-catenin (Cell Signaling Technology), Runx2 (Cell Signaling Technology), or RNAP-II (Millipore, Billerica, MA, USA) using the manufacturer’s recommendations.
For some of the experimental studies, the cells were washed and incubated with Wnt3a (100 ng/mL) or Bmp2 (100 ng/mL) for 4 hours. About 45 minutes before stimulation, some of the cells were incubated with TNFα (10 ng/mL). After stimulation, protein synthesis was inhibited by incubating the cells with cycloheximide for 3 hours, after which ChIP assay was carried out. The chromatin was immunoprecipitated using antibodies for p65 (Active Motif), p50 (Cell Signaling Technology), HDAC1 (Active Motif), β-catenin (Cell Signaling Technology), Runx2 (Cell Signaling Technology), or RNAP-II (Millipore, Billerica, MA, USA) using the manufacturer’s recommendations.