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Western blot analysis

NI Naoki Ishiuchi
AN Ayumu Nakashima
SD Shigehiro Doi
RK Ryo Kanai
SM Satoshi Maeda
ST Shinya Takahashi
MN Masataka Nagao
TM Takao Masaki
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Sample collection and western blotting were performed as described previously [20]. Rabbit polyclonal anti-Sirt1 antibody (Sigma-Aldrich), rabbit monoclonal anti-p16INK4a antibody (Abcam, Cambridge, UK), mouse monoclonal anti-GAPDH antibody (Sigma-Aldrich), mouse monoclonal anti-α-SMA antibody (Sigma-Aldrich), mouse monoclonal anti-TGF-β1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit monoclonal anti-phosphorylated Smad2 (p-Smad2) antibody (Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-Smad2 antibody (Cell Signaling Technology), mouse monoclonal anti-α-tubulin antibody (Sigma-Aldrich), rabbit monoclonal anti-CD163 antibody (Abcam), and rabbit polyclonal anti-CD68 antibody (Abcam) were used as primary antibodies. Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Dako, Glostrup, Denmark) or goat anti-mouse immunoglobulin G (Dako) were used as secondary antibodies. SuperSignal West Dura or Pico system (Thermo Fisher Scientific, Rockford, IL, USA) was used to detect signals. The intensity of each band was analyzed by ImageJ software (version 1.47v; National Institutes of Health) and standardized by the level of either GAPDH or α-tubulin.

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