Anti phospho smad 2 3

Tissue samples and cells were homogenized on ice in RIPA buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS) containing complete mini and phosSTOP solution (Roche, Mannheim, Germany). The mixture was centrifuged at 15,000xg. for 30 min and the supernatant was subjected to SDS–PAGE. Protein concentrations were determined by the Bradford method using a colorimetric assay (Bio-Rad, Hercules, CA). Western blot analysis was performed according to standard procedures using the following primary antibodies: anti-phospho Smad 2/3 (Ser 423/425; Santa Cruz), anti-Smad 2/3 (Cell Signaling Technology, #3102), anti-phospho Smad1 (Cell Signaling Technology, #9553), anti-Smad1 (Cell Signaling Technology, #9743), and GAPDH (Cell Signaling Technology). Antigens were revealed by Immobilon Western HRP Substrate (Millipore, Billerica, MA) after incubation with horseradish peroxidase-conjugated secondary antibody. The band’s density was quantified using ImageJ software.

NPDFs were lysed using PRO-PREP^TM protein extraction solution (iNtRON Biotechnology, Seongnam, Korea). Cell lysates were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore Inc., Billerica, MA). Membranes were probed with primary antibodies overnight at 4 °C. The primary antibodies used included the followings: anti-α-SMA (Chemicon, Millipore Inc., Billerica, MA), anti-acetyl H3 (Cell Signaling), anti-histone H3, anti-fibronectin, anti-phospho-smad2/3, anti-total smad2/3, and glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Membranes were washed for 5 minutes three times, incubated with peroxidase-conjugated anti-mouse or anti-rabbit antibodies (Vector Laboratories, Burlingame, CA) for 1 hour, and washed for 5 minutes three times. Protein expression was detected using Amersham ECL Western Blotting Substrate (GE Healthcare, Little Chalfont, Buckinghamshire, UK).