TGFβ2- and BSA-treated cell aggregates were washed in Hanks’ balanced salt solution and then lysed in 2× sample buffer (0.5 mM Tris-HCl, 10% SDS, 2-mercaptoethanol, glycerol, pH 6.8). Cell lysates were then boiled at 95°C for 5 min and incubated on ice for another 5 min. Each protein sample (8 μl/lane) was run on 12% SDS-PAGE and transferred electrophoretically to an Immobilon-P transfer membrane (Millipore, Bedford, MA, USA). The blots were incubated with 5% skim milk in T-TBS (50 mM Tris-HCl, 100 mM NaCl, 0.1% Tween 20, pH 7.4) for 1 hr and probed overnight with rabbit polyclonal anti-fibromodulin antibody (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in Can Get Signal Solution (Toyobo, Osaka, Japan), followed by incubation for 1 hr with horseradish peroxidase (HRP)-labeled secondary antibodies (Envision+System-HRP, anti-rabbit, Dako). Anti-β-actin antibody (0.1 μg/ml; BioVision. Mountain View, CA, USA) was used as a control.
Immunoreactive bands were visualized by ECL plus Western Blotting Detection Reagents (GE Healthcare,Mississauga , ON, Canada) with an LAS-3000 imaging system (Fujifilm, Tokyo, Japan). The acquired images were analyzed by densitometry using ImageJ software (NIH, Bethesda, MD, USA). The data were normalized with β-actin.
Immunoreactive bands were visualized by ECL plus Western Blotting Detection Reagents (GE Healthcare,