Pro pre protein extraction solution

Cells or tissues were lysed in PRO-PRE Protein Extraction Solution (Intron Biotechnology, Seongnam, Korea). Whole cell lysates were prepared from tumor cell lines or tumor tissues and cells were lysed with lysis buffer containing 1 mM EDTA, 1% TX-100, 100 mM NaCl, 50 mM NaF, TrisHCl pH 7. 5, phosphatase inhibitor cocktail (Sigma-Ald rich, St. Louis, MO). Protein concentration was determined by Bradford assay kit (BIO-RAD, Hercules, USA). Cell or tissue lysates (40 μg of total protein) were separated in 8% acrylamide gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Hybond-ECL nitrocellulose filter paper (Amersham Biosciences, Buckinghamshire, UK). Membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 for 1 hour at room temperature. Protein bands were probed with antibodies against c-MET and phospho-c-MET (p-c-MET) at a 1:200 dilution (Abcam ab74217 & ab5662), or total-Akt, phospho-Akt (p-Akt), total-ERK, phospho-ERK (p-ERK) (Cell Signaling Technology), and β-actin at 1:3000 (Santa Cruz, USA), and then labeled with horseradish peroxidase-conjugated anti-rabbit antibody (GE Healthcare, Piscataway, USA). Bands were visualized by enhanced chemiluminescence using an ECL kit (Amersham Biosciences, Buckinghamshire, UK) in accordance with the manufacturer’s protocol.

Whole cell extraction was conducted using PRO-PRE Protein Extraction Solution (Intron Biotechnology, Seongnam, Korea). Equal amounts (20 μg) of each sample were separated on 8-15% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.5) for 1 hr at room temperature, washed with TBST, and subsequently incubated with primary antibodies: anti-S100A14 (Proteintech, Chicago, IL), anti-phospho-Akt (ser473), anti-Akt, anti-phospho-Erk1/2, anti-Erk1/2 (Cell Signaling, Danvers, MA), anti-TP53 (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-GAPDH (Santa Cruz Biotechnology). Primary antibodies against each protein were detected by secondary antibodies conjugated with horseradish peroxidase (GE Healthcare, Munich, Germany). Specific bands for each protein were detected on AGFA X-ray film (Agfa Health Care, Mortsel, Belgium) using the SuperSignal Chemiluminescence kit (Thermo Scientific, Rockford, IL).

For the analysis of active caspase-3 and caspase-8 expression, cells were lysed in PRO-PRE Protein Extraction Solution (Intron Biotechnology, Korea). The protein concentration was determined using a BCA protein kit (Thermo Scientific, USA). The concentrations of active caspase-3 and caspase-8 (Invitrogen, USA) were determined using an Enzyme-linked immunosorbent assay (ELISA) kits used according to the manufacturer’s instructions. All samples were measured in triplicate.

Cells were lysed in PRO-PRE-Protein Extraction Solution (Intron Biotechnology, Seongnam, Korea). Protein concentrations were determined using a Bradford assay kit (BIO-RAD, Hercules, CA, USA). Cell lysates (50 μg of total protein) were separated in 8% acrylamide gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Hybond-ECL nitrocellulose filter paper (Amersham Biosciences, Buckinghamshire, UK). Membranes were blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature. Protein bands were probed with VEGFR2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1:500 dilution; phospho-VEGFR2 (Tyr951, Tyr1175), total-ERK (Thr202/Tyr204), phosphor-ERK (Thr202/Tyr204), total-AKT, phospho-AKT total-p38, phospho-p38 (Cell Signaling, USA) at 1:1000 dilutions; β-actin antibody at a 1:4000 dilution (Santa Cruz Biotechnology, Santa Cruz, USA) and then labeled with horseradish peroxidase-conjugated anti-rabbit antibody (GE Healthcare, Piscataway, USA). Bands were visualized by enhanced chemiluminescence using an ECL kit (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer’s protocol, as described previously^{23 (link)}.