Blot stripping buffer

Equal amounts of total protein (10–20 μg) were subjected to SDS-PAGE using 10 to 15% acrylamide gels, transferred to a polyvinylidene difluoride (PVDF) membrane, and then incubated overnight at 4°C with primary caspase antibodies (Cell signaling) and a phospho-mixed lineage kinase domain-like (MLKL) antibody (Cell signaling). The membrane was incubated with peroxidase-conjugated secondary antibodies at room temperature for 1 hour, and then proteins were visualized using enhanced chemiluminescence (Amersham, Piscataway, NJ). The blot was stripped with Blot Stripping Buffer (Thermo Fisher Scientific), and then re-probed with other antibodies as needed for the experiment.

A total of 100 prophase-I arrested oocytes were pooled and mixed with Laemmli sample buffer (Bio-Rad, cat #161–0737) and denatured at 95°C for 10 min. Proteins were separated by electrophoresis in 10% SDS polyacrylamide precast gels (Bio-Rad, #456–1036). The separated polypeptides were transferred to nitrocellulose membranes (Bio-Rad, #170–4156) using a Trans-Blot Turbo Transfer System (Bio-Rad) and then blocked with 2% ECL blocking (Amersham, #RPN418) solution in TBS-T (Tris-buffered saline with 0.1% Tween 20) for at least 1h. The membranes were incubated overnight using the antibody dilution anti-AURKA (1:500; Bethyl #A300-072A), or 1 h with anti-MSY2 (1:20,000; gift from R. Schultz) as a loading control. After washing with TBS-T five times, the membranes were incubated with anti-rabbit secondary antibody (1:1000; Kindle Bioscience, #R1006) for 1 h followed with washing with TBS-T five times. The signals were detected using the ECL Select western blotting detection reagents (Kindle Bioscience, #R1002) following the manufacturers protocol. Membranes were stripped prior to loading control detection using Blot Stripping Buffer (ThermoFisher Scientific #46430) for 30 minutes at room temperature.