Rabbit anti gapdh

All liver and ovary tissues were homogenized with LN2 and lysed on ice with RIPA buffer (SIGMA-Aldrich) containing protease inhibitor cocktail (Roche Diagnostics) and a phosphate inhibitor (A.G Scientific, Inc.). Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride membranes (PVDF; Bio-Rad). The membrane is blocked with 5% BSA (Amresco) for 1 hour at RT, and then incubated with primary antibodies for overnight at 4° C. Antibodies were used in this study included rabbit anti-NOX4 (1:1000, Abcam), mouse anti-HO-1 (1:1000, Novus) rabbit anti-Catalase (1:1000, Abcam), mouse anti-SOD1 (1:1000, Cell Signaling), rabbit anti-albumin (1:500, Novus), mouse anti-Nobox (1:500, Santa Cruz), rabbit anti-Nanos3 (1:1000, Abcam), goat anti-Lhx8 (1:500, Santa Cruz), rabbit anti-GAPDH (1:1000, Abfrontier) and horseradish peroxidase – (HRP-) conjugated secondary antibody (anti-rabbit IgG; 1:10000; Cell signaling and anti-mouse IgG antibody; 1:5000, Cell Signaling). All experiments were performed in triplicate. Intensity of each band was quantified by Image J software (NIH, Bethesda, http://www.nih.gov).

The liver tissues and cells were lysed in lysis buffer (Sigma-Aldrich) supplemented with Phosphatase Inhibitor Cocktail II (A. G Scientific Inc., San Diego, CA, USA) and Complete Mini Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Total proteins were loaded onto SDS-PAGE gels and transferred to PVDF membranes (BIO-RAD, Hercules, CA, USA), and then incubated overnight at 4 °C with one of the following primary antibodies as indicated: rabbit anti-Col Ⅰ) (1:1000; Novus, Centennial, CO, USA), mouse anti-α-SMA (1:1000; Dako), mouse anti-VEGF (1:500; Novus), rabbit anti-ALB (1:1000; Novus), mouse anti-Cyclin D1 (1:1000; Abfrontier, Seoul, Korea), rabbit anti-hepatic nuclear factor α (HNF1α; 1:1000; Abcam Inc., Cambridge, MA, USA), mouse anti-IL-6 (1:1000; Abcam), rabbit anti-glycoprotein 130 (gp130; 1:250; Santa Cruz Biotechnology), rabbit anti-phosphorylated Stat3 (1:500; Cell Signaling), mouse anti-Stat3 (1:500; Cell Signaling), and rabbit anti-GAPDH (1:3000; Abfrontier). The membranes were washed and then reacted with a secondary antibody (horseradish peroxidase-conjugated anti-mouse IgG (1:5000; Cell Signaling) or anti-rabbit IgG (1:10,000; Cell Signaling) for 1 h at RT. The membranes were incubated using enhanced chemiluminescence reagents (Thermo Fisher Scientific., Waltham, MA, USA).

The isolated hippocampus and cortex samples were homogenized in buffer containing 20 mM Tris-HCl, pH 7.0, 6 M urea, 150 mM NaCl, 2 mM EDTA, and 1% Triton X-100. The homogenates were subjected to 8% SDS-polyacrylamide gel electrophoresis and analyzed by Western blot using rabbit anti-TCTP (diluted 1 : 1000, Abcam, Cambridge, MA), rabbit anti-SUCLA2 (diluted 1 : 1000, Abcam), rabbit anti-NSE (diluted 1 : 1000, Abcam), and rabbit anti-GAPDH (diluted 1 : 10,000, AbFrontier, Seoul, Korea) antibodies at 4°C overnight. The membranes were incubated with the indicated secondary antibody (diluted 1 : 5000, GE Healthcare, Madison, WI). All values were corrected with reference to the value for GAPDH, used as an internal standard. Immunoreactivity was detected by using an Amersham ECL Prime Western blotting detection kit (GE Healthcare). Western blot images were quantified using the Multi Gauge version 2.2 software (Fuji Photofilm, Tokyo, Japan).