Anti aurka

Oocytes (n=50 per group) were frozen in RIPA buffer supplemented with a protease and phosphatase inhibitor solution. The samples were thawed on ice and mixed with 5x loading buffer, then heated at 98°C for 7 min. Proteins were separated in 10% acrylamide gels, then transferred onto a hydrophobic PVDF membranes (Milipore, Burlington, MA). The membrane was blocked in TBST supplemented with 2% Tween-20 and 5% FBS for 1h at room temperature, and incubated with anti-pT288 AURKA (1/1000; Cell Signaling Technology, Beverly, MA) at 4°C overnight. The following day, the membrane was washed in TBST and incubated with a peroxide-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) for 1h. ECL (Millipore) was used for chemiluminescent detection. The membrane was also probed with anti-AURKA (1/1000; Novus Biologicals) and anti-β tubulin (1/2000; Sigma Aldrich) as an internal control, under similar conditions. Individual band intensity was quantified using the ImageJ software and the relative total protein values in each group were compared to the control, which was normalized to 1.0.