Pcna rabbit pab

DME/F-12 cell culture medium (HyClone, Logan, UT, USA), Fetal bovine serum (HyClone, Logan, UT, USA), Trypsin (HyClone, Logan, UT, USA), FSHR immunohistochemical kit (anti-rabbit) (Xuanya Bio, Shanghai, China), PCNA Rabbit pAb (Bioworld, St. Louis, MN, USA), β-Actin Rabbit pAb (Proteintech, Wuhan, China), Anti-Rabbit IgG (H+L) (Proteintech, Wuhan, China), BeyoClick™ EdU-594 (Beyotime, Shanghai, China), Recombinant Human BMP-6 (R&D Systems, Minneapolis, MN, USA), Annexin V-FITC/PI, Protein Marker (Solarbio, Beijing, China), RNAiso, Prime Script™ RT reagent Kit with gDNA Eraser, and TB Green™ Premix Ex Taq™ Ⅲ (TaKaRa, Kusatsu, Japan).

According to the instructions of the animal whole protein extraction kit (Solarbio, Shanghai, China), the total protein of cells with different treatments was extracted after the cells were treated. The total protein was subpackaged as required and stored in a −80 °C refrigerator (Sanyo, Osaka, Japan). The protein concentration was determined using a BCA protein assay kit (Bioground, Chongqing, China). Approximately 20 µg of protein samples per well were separated on 12% SDS PAGE gels and blotted on a polyvinylidene fluoride (PVDF) membrane (Biosharp, Beijing, China). The proteins were blocked in TBST containing 5% non-fat dry milk with slight shaking for 1 h at room temperature and then washed three times for 10 min with TBST buffer (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20). Then, the membranes were incubated overnight at 4 °C with primary antibodies PCNA Rabbit pAb (1:1000; Bioworld, St. Louis, MN, USA) and β-Actin Rabbit pAb (1:5000; Proteintech, Wuhan, China). After washing with TBST buffer, the membranes were incubated with secondary antibody anti-rabbit (1:2000; Proteintech, Wuhan, China) at room temperature for 2 h. After washing at least three times, the protein bands were visualized with ECL (Bioground, Chongqing, China).