Mmp 2

The cells were harvested, washed twice with cold phosphate-buffered saline (PBS) and lysed in buffer [50 M Tris-HCl (pH 7.4), 150 M NaCl, 2 M EDTA and 1% NP-40], containing protease inhibitors. The protein concentration was quantified using a bicinchoninic acid protein measurement kit (Shenneng Bocai Biology, Co., Ltd., Shanghai, China). A total of 30 μg extract was subjected to SDS-PAGE and then electrophoretically transferred to a nitrocellulose membrane, which was blocked with 5% (w/v) dried skimmed milk-tris-buffered saline and tween 20 [TBST; 10 M Tris-HCl (pH 8.0), 150 M NaCl and 0.05% Tween 20] for 1 h at room temperature. Subsequently, the membrane was incubated for 2 h with antibodies against RECK (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, USA; at 1:1000 dilution) and MMP-2 (Santa Cruz Biotechnologies, Inc.; at 1:1000 dilution), respectively, in TBST with 5% skimmed milk. Subsequent to being washed in TBST, horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, Inc.) was used as a secondary antibody (1:10,000, in TBST with 2% bovine serum albumin incubated for 1 h). Each sample was also probed with an anti-GAPDH antibody (Santa Cruz Biotechnology, Inc.) as a loading control. Protein bands were visualized by the Alpha Imager 2200 system (Alpha Innotech, San Leandro, CA, USA).

A standard Western blotting protocol was used as described previously [5 (link),25 (link)] with antibodies against TGFβ (1:600), matrix metalloproteinase 2 (MMP2, 1:200) (Santa Cruz, CA, USA); connective tissue growth factor (CTGF, 1:500) (Biovision, Mountain View, CA, USA), LOX-1 (1:1000) (R&D System), bone morphogenetic protein-7 (BMP-7, 1:5000) (AbDSerotec, Minneapolis, MN, USA), nuclear factor-kappa B (NFκB, 1:1000), its inhibitor kappa B alpha (IκBα, 1:200), Phospho-P38 (1:1000) (Millipore, Billerica, MA, USA), NADP(H) oxidase subunit Gp91phox (1:500, BD Transduction Laboratories, France). Loading controls were β actin (1:3000, Sigma Aldrich, France) or P38 (1:1000, Millipore). Appropriate HRP-coupled secondary antibodies (1:5000 to 1:10 000, GE Healthcare, France) were used to detect the band by chemiluminescence with ECL plus (GE Healthcare, France). Intensities of the protein bands were determined and quantified using AlphaEase FC software (Alpha Innotech Corporation, San Leandro, CA).

Total protein (equal amounts) were separated by SDS-PAGE, transferred onto Immobilon®-P Transfer Membrane (Millipore Corporation, Billerica, MA, USA) and blocked with 5% non-fat milk. The membranes were incubated with the respective primary antibodies against Mmp2 (Santa Cruz, CA, USA), Mmp9 (Santa Cruz, CA, USA), Bcl-2 (Santa Cruz, CA, USA), Bax (Santa Cruz, CA, USA), pro-caspase-3 (Cell Signaling Technology, Danvers, MA, United States), cleaved-caspase-3 (Cell Signaling Technology, Danvers, MA, United States), caspase-9 (Santa Cruz, CA, USA) and β-actin (Santa Cruz, CA, USA), and then incubated with the appropriate concentrations of horseradish peroxidase-conjugated secondary antibody. The blots were visualized using the SuperSignal West Pico Chemiluminescent Substrate® (Thermo Scientific, Rockford, IL, USA).

Total proteins were extracted using 100 μl lysis buffer form cells. After measurement of the quality and concentrations of the proteins, 25 μg samples were loaded and separated in 10% sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose membranes through electroblotting. Then membranes were blocked with 5% skim milk for 1 h at room temperature, followed by incubation with P-gp (Cat. No. MA5-13854, Invitrogen, Carlsbad, CA, USA), MRP1 (SC-18835), Survivin (sc-17779), MMP2 (sc-13594), pSer473-Akt (sc-293125), Akt1 (sc-5298), p-ERK (sc-7383), ERK (sc-514302), pSer33/37-β-catenin (sc-57535), β-catenin (sc-7963), p-NFκB (sc-271908), NFκB (sc-8414), p-mTOR (sc-293089), mTOR (sc-517464), and GAPDH (SC-47724) antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4 °C in a dilution of 1:1000. Membranes were washed three times with phosphate-buffered saline Tween-20 and incubated with HRP-conjugated secondary antibody (Merck & Co., Inc., Kenilworth, NJ, USA) for another 1 h in a dilution of 1:2500. Immunoreactivity was detected using the Western Lighting Ultra (Thermo Fisher Scientific, Waltham, MA, USA).