Ab192241

Western blotting analyses were conducted as previously described (Su et al., 2001 (link)). Briefly, total protein was extracted in MEM-R, according to the manufacturer's protocol (Pierce, Rockford, USA). The nuclear and cytoplasmic proteins from fetal ovaries were extracted using a Nuclear-Cytosol Extraction Kit (Applygen Technologies Inc., Beijing, China). Protein concentrations were measured by a BCA assay procedure (CellChip Beijing Biotechnology Company, Beijing, China). The samples were separated on 15% SDS–PAGE and then transferred to Protran nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany), which were incubated with appropriate primary antibodies overnight at 4°C. Antibody against CYP51, H3.1 (#21137-1, Signalway Antibody, USA), REC8 (#ab192241, Abcam), STAG3 and MSY2 were used at dilutions of 1:500, 1:100, 1:500, 1:400 and 1:500, respectively. The appropriate peroxidase-conjugated secondary antibodies (#ZB2301, ZSGB-BIO, Beijing, China) were diluted 1:5000 in TBST. The membranes were visualized using an enhanced chemiluminescence detection system (Amersham, Arlington Heights, USA). GAPDH was used as an internal control for total protein detection, while H3.1 was used as an internal control to normalize nuclear protein expression.