Anti p p65 antibody

Anti-p-p65 antibody, Gö6976, heparin, PMA, Röttlerin, and PKC-δ siRNA were purchased from Sigma-Aldrich (St.Louis, MO). Bay 11-7082, Bay 61-3606, piceatannol, and caffeic acid phenethyl ester (CAPE) were purchased from Cayman Chemicals (Ann Arbor, MI). Lipofectamine 3000 reagent was acquired from Invitrogen Life Technologies (Gaithersburg, MD). Antibody specific for Thy-1, and p-PKC-δ was purchased from Cell Signaling Technology (Danvers, MA). Anti-Syk, p65, PKC-δ, and G3PDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody specific for p-Syk was purchased from EMD Millipore (Temecula, CA). Dual-Luciferase assay kit was purchased from Promega (Madison, WI). Penicillin and streptomycin were purchased from HyClone (South Logan, Utah). Endothelium cell growth factor (ECGS) was purchased from Biomedical Technologies (Stoughton, MA). Fetal calf serum (FCS), Medium 199 (M199), and GlutaMAX Supplement were obtained from GIBCO (Grand Island, NY). Endothelial Cell Growth Medium MV was purchased from PromoCell (Heidelberg, Germany).

Sections of surgically removed pancreatic tissue were prepared by formaldehyde fixation and cut out at 5 μm thickness from paraffin-embedded blocks. After deparaffinization with xylene, the specimens were completely dehydrated with anhydrous ethanol. After washing with ultrapure water, the specimens were immersed in sodium citrate buffer (pH 6.0) and boiled for 10 min for antigen activation. After 30 min at room temperature, the cells were washed with ultrapure water and treated with 3% hydrogen peroxide for 5 min to inactivate endogenous enzymes. Anti-p-p65 antibody (Sigma-Aldrich), anti-p-STAT3 antibody (Cell Signaling Technology), and anti-GGT1 antibody (Sigma-Aldrich) were used as primary antibodies, and biotinylated anti-rabbit IgG monoclonal antibody was used as a secondary antibody. DAB (ImmPACTTM DAB Peroxidase Substrate Kit, Vector Laboratories) was used as the chromogenic substrate.