Sc 146

Brain tissue were washed in phosphate-buffered saline once and sonicated in lysis buffer (20 mM Tris pH 7.6, 100 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1% deoxycholic acid, 10% glycerol, 1 mM EDTA, 1 mM NaVO3, 50 mM NaF, Protease Inhibitor Cocktail Set I from Calbiochem). Soluble protein was then obtained by centrifugation at 13,000 × g at 4°C for 15 minutes. Bicinchoninic acid (BCA) protein assay (Pierce) was used to measure total protein concentration. Equal amounts of lysate (20 μg protein/lane) were loaded to SDS-polyacrylamide electrophoresis on Novex Trisglycine precast gels (Invitrogen), and separated proteins were then electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore). SuperSignal West Pico Chemiluminescent Substrate system (Thermo Scientific) was applied to visualize different proteins by reacting to various antibodies. The band intensity was analyzed using Scion Image software (Scion). Antibodies used for Western blot included anti-tumor transforming growth factor beta1 (TGF-β1) polyclonal antibody (Santa Cruz, SC-146); anti-nuclear factor-kappa B (NFkB) antibody (Abcam, ab7970) and anti-actin (1:5000, Santa Cruz Biotechnology).