Anti traf6 antibody

RANKL was purchased from Peprotech (London, UK). Alpha-minimum essential media (α-MEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S) and Dulbecco’s phosphate buffered saline (DPBS) were obtained from Gibco (Gaithersburg, NY, USA). TRAP assay kit was obtained from Sigma Aldrich (Saint Louis, MO, USA). Osteo assay surface multiple well plates were obtained from Corning, Inc. (New York, NY, USA). Anti-c-Fos antibody, anti-TRAF6 antibody and anti-β-actin antibody were obtained from Santa Cruz (CA, USA). Anti-NFATc1 antibody was purchased from BD Pharmingen (San Diego, CA, USA). Anti-MMP-9 antibody and anti-CTK antibody were purchased from Abcam (Cambridge, MA, USA). Anti-total-ERK antibody, anti-phospho ERK antibody, Anti-total-JNK antibody, anti-phospho JNK antibody, Anti-total-p38 antibody and anti-phospho p38 antibody were purchased from Cell signaling (Beverly, MA, USA). Anti-NFATc1 antibody was purchased from BD Pharmingen (San Diego, CA, USA).PCR primers were obtained from Genotech (Daejeon, Korea). All of the chemicals used in the experiments were of analytical grade or complied with the level required for cell culture.

Cellular proteins were extracted and incubated with 2 μg of anti-TRAF6 antibody (1:100; Santa Cruz Biotechnology, sc-8409) overnight at 4 °C, followed by incubation with 30 μl protein A/G agarose beads (Santa Cruz Biotechnology, sc-2003) overnight at 4 °C, as described previously. The precipitations were then washed three times with lysis washing buffer and added with loading buffer. After SDS-PAGE, the membrane was subjected to western blot with anti-ubiquitin antibody (1:1000; Cell signaling technology, 5621).