For western blotting, cells were extracted with lysis buffer [20 mM Tris-HCl (pH 7.4), 1% Triton, 10% glycerol and 0.1 mM phenylmethylsulfonyl fluoride] and phosphatase inhibitors (2 mM sodium fluoride, 2 mM sodium pyrophosphate, 1 mM sodium orthovanadate). Equal amounts of protein samples (40 g) were electrophoresed on NuPAGE sodium dodecyl sulphatepolyacrylamide gels (Invitrogen), and then transferred onto Immobilon-P membranes (Millipore Corporation, Bedford). After nonspecific protein binding was blocked with skim milk and bovine serum albumin, the membrane was incubated overnight at TGF-b 1 ELISA TGF-b 1 ELISA kit specific for human TGF-b 1 (TGF-b 1 ELISA, IBL, Flughafenstrasse, Hamburg) was used according to the manufacturer's protocol. Briefly, 100 ml of samples was added to the TGF-b 1 mAb pre-coated plate and incubated overnight at 4uC. After washing, 100 ml of antiserum was added and incubated for 2 hours at room temperature for blocking. After washing, antimouse IgG antibody conjugated to biotin was added and incubated for 45 minutes at room temperature. The color was developed with 100 ml of streptavidin peroxidase and substrate.