Ab23980

Chondrocytes were induced from ATDC5 cell line for 7 days and Chromatin immunoprecipitation (ChIP) was performed as previously described [22 (link)]. After immunoprecipitation using monoclonal anti-Runx1 antibody (ab23980; Abcam) and DNA extraction, quantitative PCR was performed using the primers in the promoter region of the mouse gene Ihh. Primer sequences are presented in the Supplementary Table S2.

Anti-Flag mAb (M2, Sigma-Aldrich) were used for immunoprecipitation. Protein extracts from Myc-Flag-tagged PU.1-infected Scid.adh.2c2 cells were prepared using RIPA buffer (1% NP- 40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 µg/ml of aprotinin, leupeptin and pepstatin, 1 mM Na₃VO₄, 1 mM NaF and Tris-HCl, 50mM, pH 7.4). For immunoprecipitation analyses, the cell lysates were subjected to a pre-clear process with control IgG coupled agarose (Sigma-Aldrich) at 4^oC for 1 hr with rotation. The pre-cleared extracts were subjected to immunoprecipitation with an anti-Flag M2 agarose (Sigma-Aldrich) at 4^oC for 16 hr, and then the immunocomplexes were eluted from the agarose by 3xFlag peptide, and the eluted PU.1 complexes were run on 10% polyacrylamide gels. After electrophoresis, the proteins were subjected to immunoblotting as described previously (Hosokawa et al., 2013 (link)). Nuclear extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). The antibodies used for the Immunoblot analyses were anti-Satb1 (ab109122, Abcam), anti-Runx1 (ab23980, Abcam), anti-Lamin B (sc-6217, Santa Cruz Biotechnology, Inc.), and anti-Myc (PL14, MBL).