Enhanced chemiluminescence light method

Protein extracts were run on 10–12% SDS acrylamide gels and transferred onto nitrocellulose as previously described [19 (link)–21 (link)]. Blots were incubated with anti-caspase-3, anti-PARP, anti-phospho-Erk, anti-phospho(S536)-p65, anti-phospho(S473)-Akt (all Cell Signaling Technology, Beverly, MA), anti-IκBα, anti-phospho(S63)-c-Jun (both Santa Cruz Biotechnology, Santa Cruz, CA), anti-α-smooth muscle actin (αSMA, Sigma-Aldrich, St. Louis, MO) overnight at 4°C. After incubation with secondary horseradish-peroxidase conjugated antibodies (Santa Cruz Biotechnology), the bands were visualized by the enhanced chemiluminescence light method (Amersham Biosciences) and exposed to X-omat film (Eastman Kodak Co., New Haven, CT) or a chemiluminescence imager (Image Station 2000R, Eastman Kodak Co.). Blots were reprobed with monoclonal anti-actin (MP Biomedicals) or GAPDH (Cell Signaling Technology) to demonstrate equal loading.

Protein extracts were run on SDS acrylamide gels and transferred onto nitrocellulose. Blots were incubated with anti-Tβ4, PDK1, Akt, phospho-Akt (Ser473), P70S6, α-SMA, Vimentin, GFAP, and GAPDH (Cell Signaling Technology, Beverly, MA, USA) overnight at 4°C. After incubation with secondary horseradish-peroxidase conjugated antibodies (Santa Cruz Biotechnology), the bands were visualized by the enhanced chemiluminescence light method (Amersham Biosciences) and exposed to X-omat film (Eastman Kodak Co., New Haven, CT) or a chemiluminescence imager (Image Station 2000R, Eastman Kodak Co.).