Ten embryos were lysed in modified Barth solution (MBS) (88 mM NaCl, 1 mM KCl, 0.7 mM CaCl2, 1 mM MgSO4, 2.5 mM NaHCO3, 5 mM HEPES, pH 7.4), 1% Triton X-100, 5 mM EDTA and proteinase inhibitors for 30 min on ice and centrifuged at 10,000 g for 30 min at 4°C. The protein content was measured using the Bradford method (Bio-Rad), with BSA as the standard. Equal protein amounts were separated on 8% SDS-PAGE, and immunoblotting was performed using an immunopurified anti-DS-epi1 antibody (1 μg/ml) (Pacheco et al., 2009a).
