Ab185141

Automatic western blots were performed using a Wes automated system (ProteinSimple, California, USA). Purified recombinant proteins were used as calibration standards. Serial dilutions of both the sample and standard were used to determine the linear dynamic range of the assay. According to the ProteinSimple kit (SM-W004), the loading order was: ladder and Experimental protein samples; antibody Diluent II; antibody Diluent II and Primary Antibody; streptavidin-HRP and Secondary HRP Conjugate; luminol-Peroxide mix. SW software was used for data generation and analysis. Antibodies against p-LRP6 (ab226758, 1:50), LRP6 (ab134146, 1:10), p-GSK3b (ab68476, 1:100), GSK3b (ab185141, 1:100), β-catenin (ab16051, 1:50), LEF1 (ab137872, 1:10), and β-actin (ab6276, 1:100) were obtained from Abcam. Antibodies against p-β-catenin (4176, 1:100) were obtained from Cell Signaling Technology.

Western blotting was performed according to a previous method [36 (link)]. Protein isolation from mouse ovaries and GCs was performed using the RIPA lysis buffer (R0020, Solarbio). A BCA protein assay kit (PC0020, Solarbio) was used to measure the protein concentration. The protein samples were denatured at 100°C for 10 min. Then, the proteins were separated by sodium sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes subsequently were blocked using 5% skimmed milk for 1 h at room temperature. After blocking, the membranes were incubated overnight with primary antibodies against CD63 (ab134045, 1 : 1000; Abcam), calnexin (ab133615, 1 : 1000; Abcam), HBP1 (ab216844, 1 : 1000; Abcam), T cell factor (TCF; ab272235, 1 : 1000; Abcam), lymphoid enhancer factor (LEF; ab137872, 1 : 1000; Abcam), p-GSK3β (ab68476, 1 : 1000; Abcam), histone H3 (ab1791, 1 : 1000; Abcam), GSK3β (ab185141, 1 : 5000; Abcam), β-catenin (ab184919, 1 : 1000; Abcam), and GAPDH (ab9485, 1 : 2500; Abcam). The next day, the membranes were washed three times using Tris-buffered saline Tween-20 (Sigma-Aldrich) and incubated with secondary antibodies at room temperature for 1 h. The protein level was detected using the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA).