P smad2 3108

Antibodies used in this study included: YAP (#14074), β actin (#4967S), pSmad2 (#3108), and Smad2 (#5339) or Smad2/3 (#5678) for pSmad2 normalization (all from Cell Signaling Technology, New England Biolabs, Ltd., Whitby, ON, Canada). TβR1 (v-22) (#sc-398) was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). TβRII (ab612143), anti-Histone H3 (phospho S10, ab5176), pSmad3 (ab52903), and Smad3 (ab28379) were purchased from Abcam. TAZ/WWTR1 antibody (HPA007415) and secondary antibody, goat anti-rabbit IgG-peroxidase (A0545), were purchased from Sigma-Aldrich (Oakville, ON, Canada). rhTGFβ1 (PHG9204) was purchased from Invitrogen, Life Technologies (Burlington, ON, Canada). LY2157299 (Cayman Chemicals) was purchased from Cedarlane (Burlington, ON, Canada). Doxorubicin (Accord Healthcare, Kirkland, QC, Canada) was obtained from the Ontario Veterinary College Pharmacy.

Fresh hepatocytes and AS-30D cells were lysed with RIPA buffer plus a cocktail of protease inhibitors (cOmplet protease Inhibitors, Roche, Basel, CHE) and phosphatase inhibitors (1 mM NaF, 1 mM NaVO3); 100 μg of total protein extracts were run in SDS-PAGE as described before (36 (link)). Antibodies used were the following ones: vimentin 5741, pSmad2 3108 (Cell Signaling Technology, Danvers, MA, USA), osteopontin AF808 (R&D Systems, Minneapolis, MN, USA), PMCA 1-4 sc-20028 (Santa Cruz Biotechnology, Dallas, TX, USA), and β-actin A2228 (Sigma-Aldrich, St. Louis MO, USA). Recombinant Human TGF-β was obtained from PeproTech (Rocky Hill, NJ, USA). AS-30D cells were cultured in DMEM with 10% FCS; cells were incubated with 2 ng/ml of TGF-β for 24 or 48 h.