Anti runx2 monoclonal antibody

ATDC5 cells were cultured as described above and incubated in 5 plates of 15-cm culture dishes. After fixation of formaldehyde solution, sonication was performed with a Covaris S2 (Covaris, Wobrum, MA, USA). Sonicated samples were enriched by immunoprecipitation with anti-RUNX2 monoclonal antibody (host: rabbit, clone: D1L7F, Cell Signaling Technology, Danvers, MA, USA) and Dynabeads Protein A (Life Technologies). Rabbit IgG (Cell Signaling Technology) was used as a control. Input and immunoprecipitated (eluted) DNAs were analyzed by qPCR using LightCycler 96 System (Roche Life Science). Primer sequences were as follows: Adamts5 promoter (-700), FW primer 5′-ACAAAGCCAAGGACTTCCC-3′ and RV primer 5′-CCACCGGTGCTTCCTG-3′; Adamts5 promoter (-1400), FW primer 5′-CATTCAGGCTCTCTCGGACT-3′ and RV primer 5′-GAAGGCCAAACAACAGTTAAAGTAA-3′; Runx2 promoter, FW primer 5′-GTCACTACCAGCCACCG-3′ and RV primer 5′-AAAACGGAGTGAGCAAATATTTGAAG-3′; gene desert in mouse chromosome 6, FW primer 5′-ACCAAGAGCAGATCACAAAGCTA-3′ and RV primer 5′-AAATTCTGCTGTGTTCCATCATTG-3′ (Supplementary Table 4).

Chondrocytes, shaved cartilage, and HEK293T cells were minced in SDS buffer. These samples and samples for IP were transferred onto polyvinylidene difluoride (PVDF) membrane after SDS-PAGE. To detect the targeted protein, we used primary antibodies as follows: anti-WWP2 polyclonal antibody (sc-11896, host: goat, Santa Cruz Biotechnology, 1:120), anti-RUNX2 monoclonal antibody (host: rabbit, clone: D1L7F, Cell Signaling Technology, 1:1000), anti-myc tag monoclonal antibody (host: mouse, clone: 9B11, Cell Signaling Technology, 1:1000), anti-myc tag monoclonal antibody (host: rabbit, clone: 71D10, Cell Signaling Technology, 1:1000), anti-DYKDDDK tag monoclonal antibody (host: mouse, clone: 9A3, Cell Signaling Technology, 1:1000), anti-HA tag polyclonal antibody (ab9110, host: rabbit, Abcam, Cambridge, MA, USA, 1:2000) and anti-GAPDH monoclonal antibody (host: rabbit, clone: 14C10, Cell Signaling Technology, 1:1000). Secondary antibody reactions and signal detection were performed using a LI-COR immunofluorescence detection system (LI-COR Biosciences, Lincoln, NE, USA). All uncropped images are provided as a Source Data File.