Phospho smad2 3101

Western blot analysis was performed as described previously [23 (link)]. Primary antibodies to α-SMA (A2547; mouse; dilution 1:1,000; Sigma Chemical Co., St Louis, MO, USA), ICAM-1 (sc-1511; goat; 1:1000) and fibronectin (sc-6953; goat; 1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), type I collagen (1310-01; goat; 1:1000; Southern Biotech, Birmingham, AL, USA), SIRT3 (5490S; rabbit), DRP1 (5391; rabbit), OPA1 (80,471; rabbit), phospho-Smad2 (3101; rabbit) and phospho-Smad3 (9520; rabbit) (1:1000; Cell Signaling Technology, Danvers, MA, USA), and Smad2/3 (07-408; rabbit; 1:1000; EMD Millipore, Billerica, MA, USA) were used. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; AP0063; rabbit; 1:2000; Bioworld Technology, Inc., St. Louis Park, MA, USA) was used as an internal control. All signals were analyzed by densitometric scanning (LAS-3000, Fuji Film, Tokyo, Japan).

Total protein was extracted with ice-cold radioimmunoprecipitation buffer (Santa Cruz) containing a protease inhibitor mixture and quantified with a Bio-Rad Protein Assay Reagent. Protein samples were then separated by SDS-PAGE and transferred onto nitrocellulose membranes (Invitrogen). Membranes were blocked with 5% skim milk in TBS-T and probed with p300/GAPDH antibodies (Santa Cruz) or antibodies from Cell Signaling Technology (Phospho-Smad2, 3101; Smad2/3, 3102; Acetylated Lysine, 9441; Acetyl-histone H3 K9/K14, 9677). Goat anti-rabbit secondary antibody conjugated to horseradish peroxidise (Bio-Rad) was subsequently used and signal was visualized with an enhanced chemiluminescence western blotting detection kit (GE Healthcare).