Tgf β1 5

The cells were scraped from the plastic, suspended in PBS containing 4 mM EDTA and washed 3fold with this solution. Collagen gel was dissolved by Clostridium histolyticum collagenase NB 4G proved grade (Serva, Heidelberg, Germany). The cells were washed 3fold with EDTA-containing PBS. The proteins were extracted with cell lysis buffer (Cell Signaling). Protein content was determined with bicinchoninic acid (Sigma). Ten µg protein were applied on Novex NuPAGE 4-12 % Bis-Tris gel (Invitrogen Life Technologies, Prague) under nonreducing conditions. The proteins were transferred to 0.2 µm Hybond nitrocellulose membrane (GE Healthcare, München, Germany). Staining with Ponceau S was used as a loading control. The membranes were incubated with antibodies to α-SMA (1A4, Sigma), cellular fibronectin (IST-9; Santa Cruz Biotechnology, Heildelberg, Germany) or MMP-2 (H-76, Santa Cruz), TGF-β1 (V, Santa Cruz), Smad2/3 (D7G7, Cell Signaling), Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4, Cell Signaling) at 4 °C overnight. The secondary antibodies were from Santa Cruz. Detection was done with Western Blotting Luminol Reagent (Santa Cruz). The blots were scanned, quantified by program QUANTITY ONE 4.6 and normalized to the respective controls.