Western blot analyses were performed as previously described [37 (link)] using the Invitrogen western blot system (Thermo Fischer, Hvidovre, Denmark). Pooled oocytes were lysed in 20 µl radioimmunoprecipitation assay (RIPA) buffer (R0278, Sigma-Aldrich, Brøndby, Denmark). Proteins separated on a NuPAGE® 4–12% Bis–Tris mine gel was transferred to a membrane blocked in 5% skim milk and incubated with primary antibody (GDF9 or BMP15, Ansh Labs, 1:5000) overnight at 4 °C. Secondary horseradish peroxidase-conjugated rabbit-anti-mouse antibody (1:1000, Sigma-Aldrich) was applied for 1 h at room temperature. The Mw of the visualized bands were calculated using the AzureSpot 2.2.170 software.
The specificity of the primary antibodies used for western blotting was tested with blocking peptides against GDF9 (BG016, Ansh Labs) and BMP15 (BB028, Ansh Labs). Human rGDF9 standard (SRP4872, Sigma Aldrich) and human rBMP15 standard (5096-BM-005, R&D systems, Abingdon, UK) were used as a positive control.
The specificity of the primary antibodies used for western blotting was tested with blocking peptides against GDF9 (BG016, Ansh Labs) and BMP15 (BB028, Ansh Labs). Human rGDF9 standard (SRP4872, Sigma Aldrich) and human rBMP15 standard (5096-BM-005, R&D systems, Abingdon, UK) were used as a positive control.
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