C fos antibody

RANKL-induced osteoclast was prepared as mentioned above. On the 7 days after osteoclast induction, cells were lysed with RIPA buffer (Pierce Biotechnology, Rockford, IL, USA) containing protease inhibitors (Roche, Hoffmann, USA). 30 μg protein samples were separated in sodium dodecyl sulfate-polyacrylamide gel, and transferred to polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA). Each membrane was incubated with primary β-actin, TRAF6, NF-κB, Lamin B, IκB-α, p-IκB-α, extracellular signal–regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, NFATc1 and c-fos antibodies (Cell Signaling, USA) overnight at 4 °C. Anti-mouse IgG was used as the secondary antibody. Immunoreactivity was detected using an enhanced chemiluminescence detection system. The experiments were carried out 3 times in triplicate measurements.

RBLs were collected as previously described [30 (link)]. Proteins were crosslinked to DNA by adding 270 µL of 37% formaldehyde to 10 mL culture medium (final concentration, 1%). After 10-min incubations at 37 °C, reactions were quenched by adding 0.125 M glycine for 5 min at room temperature. After washing with ice-cold PBS twice, fixed cells were lysed with 1 mL RIPA buffer (MedChem Express, Monmouth Junction, NJ) on ice for 10 min. After centrifugation, nuclei were sonicated (30% amplitude, 20 sets of 15-s bursts) for four rounds. Debris was removed by centrifugation at 13,000 rpm at 4 °C for 10 min; supernatants were transferred to new tubes. Lysates were precleared with protein A/G beads before being incubated with c-Fos antibodies (Cell Signaling Technology, Danvers, MA) or IgG (Santa Cruz, CA) at 4 °C overnight with rotation. The immunocomplex was washed (once each) in low salt, high salt, and LiCl solutions and washed twice in TE buffer. Bead-bound immunocomplexes were eluted with 200 µL elution buffer. To reverse histone-DNA crosslinks, samples were combined with 5 M NaCl and heated at 65 °C for 4 h. DNA product was purified in a spin column system (Omega Bio-Tek, Norcross, GA).