Anti rabbit

To obtain samples for Western Blots, brain tissue surrounding the wound (70–90 mg) as well as an equal part of the contralateral, un-operated cortex were dissected out and immediately frozen at −80 °C until use. To prepare lysates, frozen brain tissues were minced with eye scissors in ice. The samples were then homogenized in lysis buffer (1% NP-40, 50 mmol/L Tris, pH 7.5, 5 mmol/L EDTA, 1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 1 mmol/L PMSF, 10 mg/mL aprotinin, and 1 mg/mL leupeptin) and clarified by centrifuging for 20 min in a micro centrifuge at 4 °C. After determination of its protein concentration with the Bradford assay (Bio-Rad, Hercules, CA, USA), the resulting supernatant (50 μg of protein) was subjected to SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to a polyvinylidine difluoridemembrane (Millipore, Boston, MA, USA) by a transfer apparatus at 350 mA for 2.5 h. The membrane was then blocked with 5% nonfat milk and incubated with primary antibody against PHB2 (Anti-Rabbit, 1:500; Santa Cruz, CA, USA) or GAPDH (Anti-Rabbit, 1:1000; Santa Cruz, CA, USA). After incubation with an Anti-Rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody, protein was visualized using an enhanced chemiluminescence system (ECL, Pierce Company, Rockford, IL, USA).

Protein extraction was performed on frozen testicular tissue by the Trizol precipitation method. Protein concentrations were evaluated using the Bradford’s test (Bio-Rad; USA). Equal amounts of each protein (30 μg) were loaded onto 12% SDS polyacrylamide gels (PAGE) which were then transferred to PVDF membrane (Bio-Rad; USA). Blocking solutions were prepared separately for each antibody. Nrf2 (1/500, Anti-rabbit, Abcam, USA), Slc7a11 (1/500, Anti-rabbit, Abcam, USA), Gpx-4(1/1000, Anti-rabbit, Abcam, USA), p-Jnk (1/1000, Anti-mouse, Abcam, USA) and β-Actin (1/500, Anti-rabbit, Santacruz, USA) were used as primary antibodies. Polyclonal IgG-HRP goat Anti-mouse (1/5000, Dako, Denmark) and IgG-HRP goat Anti-rabbit (1/16000, Santacruz, USA) were then used as secondary antibodies. Detection was performed using the GE Amersham ECL and Western Blotting kits (Amersham; GE Health care) and autoradiography [19 (link)]. Signal quantification was performed using Image J software.

AGS and MKN45 cancer cells were infected with control (Ad-Ctrl) and GPX7 (Ad-GPX7) adenovirus particles (5 MOI) for 48 hours, then cells were lysed in the presence of proteinase inhibitor cocktail and phosphatase inhibitor (Santa Cruz Biotechnology, Dallas, TX USA). The protein concentration was determined by a Pierce BCA Protein Assay (Thermo Scientific, Rockford, IL USA) using a FLUOstar OPTIMA microplate reader (BMG Labtech, Cary, NC USA). Equal amount of proteins were loaded and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, and Western blot analysis was performed using standard protocols. The primary antibodies were: anti-GPX7 antibody (rabbit, 1:1000, Proteintech Group, Chicago, IL USA), and anti-PARP and cleaved PARP (Cell Signaling). A mouse monoclonal antibody against beta-actin (Sigma) was used as a loading control. Horseradish peroxidase-conjugated anti-mouse (1:10,000 dilution) and anti-rabbit (1:10,000 dilution) secondary antibodies were purchased from Santa Cruz and Cell Signaling, respectively. Immunoreactive protein bands were documented using a Bio-Rad ChemiDoc XRST image system (Bio-Rad).