Horseradish peroxidase conjugated rabbit anti mouse antibody

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.

DF-1 and HEK293 cells were infected with the previously generated rMVAs (MOI  =  0.1) or rChAdOx1 (MOI  =  0.1), respectively, or were mock infected. At 24 h.p.i., cells were harvested, washed in PBS containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), and lysed with RIPA Buffer (Santa Cruz Biotechnology, Dallas, TX, USA). Extracts were then sonicated for 2 min and proteins were resolved in 12% SDS-PAGE and blotted onto a nitrocellulose membrane. After a blocking step with 5% low fat dry milk in TBS Tween 20 (TBST) (blocking buffer), mouse hyperimmune serum against BTV (1:500) or α-BTV NS2 MAb 23H6 (1:500) were applied to membranes in TBST-Milk 5% and incubated overnight at 4°C. Bound antibody was detected with horseradish peroxidase-conjugated rabbit anti-mouse antibody (Sigma-Aldrich, San Louis, MO, USA) diluted in TBST-Milk 5% (1:10,000) and the ECL detection system (Amersham^TM Pharmacia Biotech, Buckinghamshire, UK).