Embryos were collected in ice-cold PBS and subsequently snap frozen in dry ice. Embryonic tissues were ground to a fine powder using a mortar and pestle on dry ice and lysed in RIPA buffer (150 M NaCl, 50 mM Tris pH 8, 5 mM EDTA, 1% NP-40 (Sigma, I8896), 0.5% sodium deoxycholate, 0.1% SDS supplemented with 1X protease (Sigma, S8820) and phosphatase inhibitors (Roche, 4906845001)). Fifteen microgrammes of protein extracts were run on a 4–12% Bis-Tris Gel NuPAGE (Invitrogen, NP0322) and transferred on a PVDF Amersham Hybond-P transfer membrane (GE Healthcare, RPN303F). The membrane was blocked with 5% milk (Dominique Dutscher, 711160) in Tris-Buffer Saline 0.2% Tween (Sigma, P9416) (TBS-T) for 1 h at room temperature and probed with specific primary antibodies overnight at 4 °C. After three washes in TBS-T, the membrane was incubated with HRP or fluorophore-conjugated secondary antibodies and revealed by chemiluminescence (Pierce ECL2 western blotting substrate, Thermo Scientific, 80196) or fluorescence (Bio-Rad, Chemidoc MP) (Table (Table22).

Antibodies used for Western blot

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