Sc 8010

RIPA buffer (pH = 8.0) including 150 mM NaCl, 0.1% SDS, 50 mM Tris-HCl and 1% NP-40 was added to the proteinase inhibitor (Roche Diagnostics, Co., Ltd., Rotkreuz, Switzerland) and used to obtain whole cell lysates. The proteins were examined using a BCA kit (Thermo Fisher) and separated by SDS-PAGE. The proteins were transferred onto PVDF membranes (0.45 µm, Millipore Corp, Billerica, MA, USA), sealed with 5% BSA for 60 min at 25°C, and hybridized at 4°C with primary antibodies to MMP2 (1:1000, MA5-14,186, Invitrogen Inc., Carlsbad, CA, USA), MMP9 (1:2000, ab58803, Abcam), Bax (1:1200, sc-7480, Santa Cruz Biotechnology), PI3K (1:1,800, sc-8010, Santa Cruz Biotechnology), p-AKT (1:1,500, ab81283, Abcam), AKT (1:1,300, sc-5298, Santa Cruz Biotechnology), and GAPDH (1:3,000, G8795, Sigma-Aldrich, St Louis, MO, USA) for one night. The blots were incubated with secondary antibodies for 60 min at 25°C. Finally, the protein bands were detected using an ECL kit (Thermo Fisher Scientific). Densitometric analysis was performed using IPP 6.0 (Image-Pro Plus 6.0).

Celecoxib was generously provided by Pharmacia Cor. (New York, NY, USA) and dissolved in DMSO (40 mM) as a stock solution in 4 °C. Cisplatin was purchased from Qilu Pharmaceutical Co., Ltd. (ShanDong province, China) and dissolved in normal saline (2 mg/ml) as a stock solution in 4 °C. Celecoxib and Cisplatin were added at various concentrations to cells in 10% fetal bovine serum (FBS)-containing DMEM. The final DMSO concentration never exceeded 0.1% (v/v) after addition to medium. Mouse monoclonal antibodies specific for PI3K p110 (sc-8010), β-catenin (sc-7963); rabbit polyclonal antibodies specific for E-cadherin (sc-7870), Trkb (sc-8316), β-actin (sc-1616R);secondary antibodies were obtained from Santa Cruz Biotechnology, Inc.(Santa Cruz, CA, USA) were purchased from Calbiochem (San Diego, CA, USA).