For negative controls,

slides were processed as above but treated with PBS, instead of the primary antibody/biotinylated secondary antibody, for 30 minutes and peroxidase-labeled streptavidin for 30 minutes. Color reaction was developed with 3, 3′-diaminobenzidine as a chromogen. Finally, the slides were counterstained with hematoxylin, dehydrated through graded alcohol, and observed under the microscope. We used the Image Analysis System for protein analysis; 5 different views were selected for each slide (400 times). Integrated optical density was used as the measurement of staining strength. Western blotting Epigenetics inhibitor Whole-cell protein extracts and nuclear protein extracts from pancreatic cancer cells were prepared with RIPA Lysis Buffer (Santa Cruz Biotechnology,

Santa check details Cruz, CA, USA) and Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA), respectively, RAAS inhibitor according to the manufacturers’ instructions. Protein concentrations were determined using an assay kit (Bio-Rad, Hercules, CA, USA). Lysates containing 100 μg of protein were mixed with loading buffer with 5% β-mercaptoethanol and heated for 5 minutes at 100°C. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes by semi-dry blotting. Membranes were incubated in blocking buffer (tris-buffered saline [TBS], 0.1% Tween 20, and 5% non-fat dry milk) for 1 hour at room temperature, followed by hybridization with anti-p-Stat3 (tyr-705) antibody (Cell Signaling Technology,

1:1000 dilution), anti-Stat3 antibody (Cell Signaling Technology, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz Biotechnology, 1:500 dilution), anti-VEGF antibody (Santa Cruz Biotechnology, 1:500 dilution) or anti β-actin antibody (Lab Vision, Fremont, CA, USA, 1:100 dilution) at 4°C overnight. After 3 washes next in TBS/0.1% Tween 20, the membranes underwent hybridization with a horseradish peroxidase-conjugated secondary antibody rabbit IgG (Santa Cruz Biotechnology, 1:5000 dilution) for 1 hour at room temperature. After 3 washes in TBS/0.1% Tween 20, signals were detected by chemiluminescence using western blotting luminol reagent (Santa Cruz Biotechnology). Invasion assay The invasion assay was performed using a specialized invasion chamber (Chemicon, Temecula, CA, USA). The inserts contained an 8-μm pore size polycarbonate membrane with a precoated thin layer of basement membrane matrix (ECMatrix). Briefly, media supplemented with 10% fetal bovine serum was poured into the lower chamber as a hemo-attractant. After reaching 60-70% subconfluence, pancreatic cancer cells were trypsinized and resuspended in DMEM (1×106 cells/ml), and 0.3 ml was re-seeded into the upper chambers. Cells were cultured in medium containing either vehicle alone (control) or indicated doses of AG490.