Rabbit anti catalase

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 tissue lysates were prepared in SDS containing sample buffer, and equal volumes of lysates were separated by 12% SDS-PAGE gel. Proteins were transferred to nitrocellulose membrane and the blots were probed with the following primary antibodies: mouse anti-SOD (1:1000, Abcam); rabbit anti-catalase (1:2500, Abcam); mouse anti-HO-1 (1:10000, Abcam), and mouse anti-α-tubulin (1:5000, Abcam). Appropriated HRP-conjugated secondary antibodies were then applied to the blots. Blots were visualized by enhanced chemiluminescence (Promega™ ECL Western Blotting Substrate) and analyzed on the Odyssey Infrared imaging system (LI-COR Biosciences) (Lincoln, NE).