“…Sample preparation, protein digestion, and chromatographic separation were performed as described previously (30). Briefly, five tubes of 20 -30 embryos were lysed (7 M urea, 2 M thiourea, 4% CHAPS (all Amersham Biosciences), 100 mM DTT (Bio-Rad), Phosphatase Inhibitor Mixture 11 (Sigma), Complete protease inhibitor (Roche Applied Science)) and homogenized (TissueLyser, Qiagen; 2 ϫ 3 min, 30 Hz), and proteins were precipitated and resuspended in 0.2% (w/v) RapiGest TM SF (Waters Co., Milford, MA) in 50 mM ammonium bicarbonate (Sigma).…”