Runx2 m70 antibody

PC3-H cells (TGFβ1 treated or not treated) were scraped into ice-cold PBS and pelleted by centrifugation. Cells were then lysed with lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1% IGEPAL CA-630, EDTA-free cOmplete protease inhibitor cocktail (Roche, Nutley, NJ), 25 mM MG132) on ice for 20 min and centrifuged to pellet insoluble cellular debris. Runx2 M70 antibody (Santa Cruz Biotechnology, Dallas, TX) or rabbit IgG (EMD Millipore, Billerica, MA) was added to supernatant and incubated at 4°C overnight with agitation. Fifty μl protein A/G PLUS Agarose beads (Santa Cruz Biotechnology, Dallas, TX) were then added and incubated at 4°C for 3 h. Protein-antibody complexes were then collected by centrifugation, beads were washed four times with lysis buffer, resuspended in 2 x SDS sample buffer (Bio-Rad Laboratories, Hercules, CA), containing 5% β-mercaptoethanol and boiled for 5 min. immunoprecipated proteins were separated by SDS-PAGE and transferred to PVDF membranes for Western blot analysis.

Chromatin immunoprecipitation from chondrogenic cells was performed using ChIP-IT kit (Active Motif, Carlsbad, CA). Briefly, cells were cross linked in 1% formaldehyde/PBS for 10 minutes at 22°C and washed first with PBS and then glycine solution. Cells were collected in PBS containing 5mM PMSF, resuspended in lysis buffer, and sonicated 4 times for 20 seconds each. Gel electrophoresis confirmed that the bulk of DNA fragments from soluble chromatin were ~ 400–500 bp in length. Immunoprecipitation was carried out for 14 hours at 4°C with 5μg of Runx2 M70 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or normal rabbit IgG as a negative control. The immunoprecipitated chromatin was collected using Protein A/G beads. Chromatin and 5% input were analyzed by real time PCR using primers designed to amplify promoter fragments spanning the RUNX motif in Sfn, Gpr132, Cyclin A1, and c-Myb genes. The primer sequences and location of RUNX binding sites in each gene are listed in Table S3. In parallel, conventional PCR was performed with 31~37 cycles of amplification. Amplicons were run on a 2% agarose gel, stained with ethidium bromide, and visualized under UV light. The cellular distribution of Runx2 protein and its interaction of Runx2 protein with target DNA were assessed by immunofluorescence and EMSA as described in supplement methods.