Enhanced chemiluminescent detection

The total protein was obtained from the PLL specimens, as described for the cytokine array above. Then, 5 μg proteins of the cell lysates were analyzed by a Western blot, using 8% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. After electrophoresis, the gels were transferred to PVDF membranes (Millipore Corp., Burlington, MA, USA) and incubated overnight at 4 °C with antibodies against the osteoprotegerin antibody (Abcam, Cambridge, UK) and runt-related homeobox (RUNX) (GeneTex, Irvine, CA, USA), followed by a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Immunoreactivity was visualized by enhanced chemiluminescent detection (Perkin Elmer Co., Waltham, MA, USA).

After experimental periods, cultured cells were washed twice with PBS and solubilized in lysis buffer containing 40 mM Tris buffer (pH 7.5), 8 M urea, 4% CHAPS, 1 mM PMSF, 1 mM Na₃VO₄, 1 mM dithiothreitol, and a protease inhibitor kit (BM, Germany). Protein was quantified by a Biorad protein assay reagent. Aliquots of proteins (5 μg) of the cell lysates were analyzed by western blot, using SDS-PAGE (8% or 12% gel), as previously described [35 (link), 41 (link)]. After electrophoresis, gels were transferred to PVDF membranes (Millipore Corp., USA) and incubated overnight at 4 °C with antibodies against PGIS (rabbit, dilution 1/3000), COX-1, COX-2 (rabbit, dilution 1/5000, Cayman) or β-actin (1:5000; Santa Cruz Biotech, USA) followed by a HRP-conjugated secondary antibody (dilution 1/2000, Jackson Lab) for 1 h at room temperature. Immunoreactivity was visualized by enhanced chemiluminescent detection (Perkin Elmer Co, USA).