Mitochondrial protein extract kit

The Annexin V-FITC Apoptosis Detection kit was purchased from KeyGEN bioTECH Company (Nan Jing, Jiang Su, China). Human Tubal Fluid (HTF) was obtained from Millipore Co., Ltd. (Bedford, MA, USA). ROS, TAC (ABTS method), and bicinchoninic acid (BCA) kits were provided by Nanjing Jiancheng Bioengineering Institute (Nan Jing, Jiang Su, China). Polyclonal anti-cytochrome C, anti-caspase-3, anti-bcl-2, and anti-bax antibodies were purchased from Cell Signaling Technology Inc. (Danvers, Massachusetts, USA). Goat anti-rabbit IgG horseradish peroxidase (HRP) conjugate and enhanced chemiluminescence (ECL) kit were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). PVDF membranes were purchased from Merck Millipore Inc. (Danvers, Massachusetts, USA). The mitochondrial protein extract kit was purchased from Beyotime Ltd. (Haimen, China). Trizol (Invitrogen, USA) was used for RNA extraction. The Ex TaqTM PCR kit was purchased from TAKARA Biotechnology Co., Ltd. (Dalian, China). SYBR Green/Flourescein qPCR Master Mix(2X) was purchased from Thermo Scientific Fermantas (USA).

Total protein was extracted by RIPA lysate containing 1% cocktail and mitochondrial protein was extracted by the mitochondrial protein extract kit (C3601, Beyotime, China). Protein was separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Then the protein was transferred onto a PVDF membrane (IPVH00010, Millipore, USA). After blocking in 5% milk for 2 h, the membranes incubated the primary antibody at 4 °C overnight. Then membranes were incubated with second antibody-HRP conjugate for 2 h at room temperature and visualized with Chemiluminescent detection reagent (WBKLS0500, Millipore, USA). Bands were analyzed using the ImageJ software (Version 1.52a, NIH).