Enhanced chemiluminescence solution

HeLa cells were lysed in 80 µL RIPA buffer (Beyotime, Shanghai, China) containing 0.8 µL 1 mM fluoride, and the protein concentration of each sample was determined using the BCA Protein Assay Kit (Bioss, Beijing, China). Proteins (20 µg) were boiled at 95 °C for 5 min, followed by separation by 15% SDS–PAGE. After electrophoresis, the proteins were electrophoretically transferred to PVDF membranes at 100 mA for 90 min. The membranes were blocked with 5% nonfat milk for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: CASP3, BCL−2, beta Actin (β−actin) (Proteintech Group, Inc., Chicago, IL, USA). The catalog numbers are 66470-2-Ig,60178-1-Ig, and 66009-1-Ig. Next, the membranes were treated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody (Proteintech Group, Inc., Chicago, IL, USA). The catalog number is SA00001-1. The membranes were visualized with enhanced chemiluminescence solution (Biosharp, Beijing, China) (A liquid:B liquid = 1:1) using an Amersham ImageQuant 800 system (Cytiva, Tokyo, Japan).

A total of 100 porcine oocytes in each group were lysed in Laemmli sample buffer and subsequently boiled at 100 °C for 10 min. The denatured proteins were separated by SDS-PAGE using 12% (w/v) gel and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After being blocked with 5% (w/v) low-fat dry milk at RT for 1 h, the membranes were incubated with primary antibodies (1:1000 for LC3A/B, BCL-2, GAPDH and α-tubulin, 1:2000 for BAX) at 4 °C for 12 h and, after five washes in TBST (Tris-buffered saline containing 0.1% (v/v) Tween 20), the PVDF membranes were incubated with an HRP-labeled secondary antibody (1:1000) at 37 °C for 1 h. After five washes in TBST, the protein bands were visualized with enhanced chemiluminescence solution (Biosharp, Hefei, China) and analyzed with the ImageJ 1.5 software.