Versican

The extracts of mouse tissue were collected using tissue lysis buffer (Beyotime, Jiangsu, China) plus 1 mmol L 1 phenylmethylsulfonyl fluoride. Total protein was quantified using a bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electropho-retically transferred to nitrocellulose membranes, which were incubated with specific antibodies for the antigens of β-actin (MBL, Nagoya, Japan), ADAMTS1 (Abcam, Cambridge, MA, USA), versican (Millipore, Temecula, CA, USA), and DPEAEE (LifeSpan BioSciences, Seattle, WA, USA), washed, and incubated with an appropriate IRDye800-conjugated second antibody (Rockland, Gilbertsville, PA, USA). Specific immunofluorescence bands were detected using the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).

Treated cells or liver fragments were washed twice in ice cold phosphate-buffered saline, then lysed and homogenized in RIPA (Boston Bio-products cat. # BP-115) supplemented with protease inhibitor cocktail tablets (Roche cat. # 11836153001). Normalized protein samples were mixed with appropriate volume of Laemmli buffer (Boston Bio-product cat. # BP-110 R), boiled for 5 min, resolved on a 10% polyacrylamide gel and immuno-blotted with specific antibodies as indicated. Protein bands were analyzed by densitometry, normalized to β-actin for each lane, using the Kodak Image Station 4000 mm Pro Chemiluminescence image analyzer and quantitated using ImageJ software (http://imagej.nih.gov/ij/). Molecular weights were estimated in comparison with Precision Plus Protein WesternC Standard (Bio-Rad cat. # 161-0376). Antibodies used included: β-actin (Abcam cat. # ab6276), histone H3 (Abcam cat. # ab1791), versican (Sigma cat. # HPA004726), versican DPEAAE neo-epitope antibody (Abcam cat. # ab19345), 55 and cleaved poly(ADP-ribose) polymerase antibody (Cell Signaling cat. # 9544). The Protein Molecular Weight Calculator tool (http://www.sciencegateway.org/tools/pro teinmw.htm) was used to calculate versican core protein fragment sizes based on known amino acid composition.