Anti total erk 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.

Anti-Eed antibody (#09-774, rabbit polyclonal, 1:1,000) was purchased from Millipore. Anti-H3K27me3 antibody (GTX1121184, rabbit polyclonal, 1:500) was purchased from GeneTex. Anti-Actin antibody (I-19, rabbit polyclonal, 1:500) was purchased from Santa Cruz Biotechnology. Anti-Hif1a antibody (NB100-449, rabbit polyclonal, 1:500) was purchased from Novus Biologicals. Anti-Bnip3 antibody (ab10433, mouse monoclonal (ANa40), 2 μg ml⁻¹) was purchased from Abcam. Anti-p-ERK1/2 antibody (#4370, rabbit monoclonal, 1:1,000), anti-p-MEK1 antibody (#9154, rabbit monoclonal, 1:1,000), anti-p-cRaf antibody (#9427, rabbit monoclonal, 1:1,000), anti-p-RSK antibody (#9335, rabbit monoclonal, 1:1,000) and anti-p-p38 antibody (#4511, rabbit monoclonal, 1:1,000), anti-total ERK antibody (#9102, rabbit polyclonal, 1:1,000), anti-p-Smad1/5/8antibody (#9511, rabbit polyclonal; 1:1,000), anti-Smad2 antibody (#5339, rabbit monoclonal, 1:1,000), anti-p-Smad2 antibody (#3104, rabbit polyclonal, 1:1,000), anti-TGF-β receptor II antibody (#3713, rabbit polyclonal, 1:1,000), anti-p-STAT1 antibody (#9171, rabbit polyclonal, 1:1,000) and anti-pSTAT3 antibody (#9145, rabbit monoclonal, 1:2,000) were purchased from Cell Signaling Technology. Western blot analysis was performed according to the standard procedure.

Proliferative signals were investigated using ERK1/2 phosphorylation, as previously described [31 (link), 32 (link)]. To these purposes, cell lines were seeded in 24-well plates (1.0x10⁵ cells/well) and treated 15 min with 1 μM 8-Br-cGMP or 50 μM vardenafil. Where needed, cells were pretreated 1 h with the NS 2028 GC inhibitor, PMA or U0126 [24 (link)]. Cells were lysed in ice-cold RIPA buffer containing protease and phosphatase inhibitors for protein extractions. The phosphorylation of ERK1/2 was evaluated using an anti-pERK1/2 primary antibody (#9101, Cell Signaling Technology Inc., Danvers, MA, USA). Lysed samples were loaded to separate proteins in a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The anti-total ERK antibody (#9102, Cell Signaling Technology Inc.) was used as a loading control. Then, membranes were incubated with a secondary anti-rabbit horseradish peroxidase (HRP)-conjugated antibody (#NA9340V; GE HealthCare).
Western blotting signals were revealed upon incubation with ECL chemiluminescent compound (GE HealthCare, Chicago, IL, USA). Images were acquired by the VersaDoc™ MP 4000 System and QuantityOne analysis software (Bio-Rad Laboratories Inc., Hercules, CA, USA).