Anti p atm

RIPA lysis buffer (Beyotime Biotechnology, China) containing 1% Halt™ Protease and Phosphatase Inhibitor Cocktail (100×) (Thermo Fisher Scientific) was used to lyse cells. Pierce BCA Protein assay kit (Thermo Fisher Scientific, USA) was used to measure the protein concentration. The protein (40 μg) was separated by 10% SDS-PAGE and then transferred to PVDF membranes (Millipore, USA). The membrane was blocked in TBST containing 3% BSA and primary antibodies were incubated overnight at 4 °C. The primary antibodies were used as follows: anti-SIRT1, anti-p53, anti-Ac-p53, anti-p-p53, anti-p-Chk2, anti-p21, anti-TRAF2, anti-p-ATM, anti-Cyclin B1 (1:1000, Abcam) and anti-GAPDH (1:5000, Huabio). Subsequently, TBST was used to wash the membranes three times and the membranes were incubated at room temperature for 1 h with 1:3000 horseradish peroxidase conjugated anti-rabbit or mouse immunoglobulin G (Southern Biotechnology Associates, USA), followed by three washings in TBST. Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare) were used to visualize the specific bands with a ChemiScope 3300 Mini equipment (CLINX, China). The same membrane loaded with GAPDH served as the control.

Oocytes were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min and permeabilized with 0.1% Triton X-100 and 0.01% Tween-20 for 20 min at room temperature. They were then blocked with 3% bovine serum albumin (BSA)-supplemented PBS for 1 h and incubated with anti-acetylated a-tubulin (1:1,000), anti-γ-H2AX (1:250, Abcam), anti-p-ATM (1:100, Abcam), or anti-MDC1 (1:250, Abcam) antibodies overnight at 4°C. After they were washed three times, the oocytes were incubated with Alexa Fluor–conjugated 488 secondary antibodies (1:500, Jackson ImmunoResearch) at room temperature for 2 h. Finally, the oocytes were counterstained with DAPI, mounted on glass slides, and observed under an LSM 700 laser scanning confocal microscope (Zeiss) with a C-Apochromat 63×/1.2 water immersion objective. To measure the fluorescence intensity, images were captured with the same laser power, and the mean intensity of the fluorescence signals was measured. Data were analyzed using ZEN 2012 Blue (Zeiss) and ImageJ software (National Institutes of Health) under the same processing parameters.
For mitochondrial staining, oocytes were incubated with Mitochondrial Staining Reagent-Red-Cytopainter (Abcam) for 1 h at 37°C and observed under confocal microscopy.