Anti rabbit igg conjugated to horse radish peroxidase

Western blotting was performed using proteins isolated from different regions of the 10 femoral heads. The bone tissues were washed twice with 0.9% NaCl and phosphate-buffered saline (PBS), and then lysed with NET-Triton Lysis Buffer (0.01 M Tris-Cl, 0.1 M NaCl, 1 mM EDTA pH 7.4, 1% Triton X-100, 10% glycerol, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, and a cocktail of protease inhibitors). Aliquots of lysates were electrophoresed and then the proteins were transferred to poly(vinylidene fluoride) (PVDF) membranes (Bio-Rad). Nonspecific binding of the antibodies to the membrane was blocked by a 1-h incubation with 5% (w/v) non-fat dry milk/0.01 (v/v) Tween 20 in Tris-buffered saline (TBS). The membranes were probed with specific antibodies against RANK (Santa-Cruz), RANKL, OPG, RUNX2, BMP2, and BMP7 (all Abcam, UK). Human β-actin monoclonal antibody (Serotec, UK) was used as a protein marker for quantification of the protein bands. Signals were detected using secondary antibodies [anti-mouse IgG and anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (dilution 1:5000, Abcam, UK)] and the membranes were immersed in ECL detection solution (Santa Cruz, USA). The protein bands were quantified using an Epson GT-8000 laser scanner. The ratios of the protein band intensities relative to that of β-actin were calculated for each sample.

Cells were harvested and lysed in Nonidet P-40 buffer containing a protease-inhibitor mixture (Sigma). Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on Mini-PROTEAN TGX Any kD Precast Gels (BioRad, Hercules, CA, USA) and subsequently transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Immunoblots from tumor cell lysates were probed with antibodies against DDX3X (Sigma), EGFR, phospho-EGFR (Tyr1068), phospho-EGFR (Tyr1173), phospho-EGFR (Tyr845), Akt, phospho-Akt, Erk1/2, phospho-Erk1/2, and β-actin (Sigma). All antibodies except for anti-DDX3X and anti-β-actin were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Secondary antibodies consisted of anti-mouse IgG (BioRad) and anti-rabbit IgG conjugated to horseradish peroxidase (Abcam, Cambridge, MA, USA). Immunoreactive protein bands were visualized using an ECL kit (Pierce). At least three independent experiments were performed for all analyses.