Lysis buffer

NPDF lysates were collected in lysis buffer (G-Biosciences, St. Louis, MO, USA) with a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The same amount of proteins was separated using 10% sodium dodecyl sulfate-polyacrylamide mini-gel electrophoresis and transferred to the nitrocellulose membrane (GE Healthcare Life Sciences, Chalfont, UK). After the night-time incubation of a specific primary antibody (α-SMA, Col-1, fibronectin, and p-Smad 2), the membrane was incubated with a secondary antibody (IgG for goat anti-mouse) conjugated to horseradish peroxidase. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system (Pierce Biotechnology, Inc., Rockford, IL, USA). Band images were captured and analyzed using image systems (AI 600, GE Healthcare Life Sciences, Canton, MA, USA) and ImageJ software (ver. 1.52a; National Institutes of Health, Bethesda, MD, USA).

NPDF lysates were collected in lysis buffer (G-Biosciences, St. Louis, MO, USA) with a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Equal amounts of protein from cell lysates were separated using 10% sodium dodecyl sulfate-polyacrylamide mini-gel electrophoresis and transferred onto nitrocellulose membranes (GE Healthcare-Life Sciences, Chalfont, UK). Following overnight incubation with the specific primary antibody (α-SMA, Col-1, fibronectin, p-Smad2 and p-Smad3), membranes were incubated with a secondary antibody (goat anti-mouse IgG) conjugated to horseradish peroxidase. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system (Pierce Biotechnology, Inc. Rockford, IL, USA). Band images were captured and analyzed using an imaging system (AI 600, GE Healthcare Life Sciences, Canton, MA, USA) and ImageJ software (ver. 1.52a; National Institutes of Health, Bethesda, MD, USA).