Flies laid on grape-agar plates for two hours and embryos were either aged two hours at room temperature or taken directly after collection. Embryos were dechorionated with bleach, rinsed, and frozen in aliquots of ~25 embryos at -80 C. Embryos were homogenized in 25 μl RIPA buffer (Sigma cat # R0278) supplemented with 1 mM DTT and protease inhibitors (Sigma cat # 4693116001) using a plastic pestle. After homogenization, samples were mixed with 25 μl 2x Laemmli buffer (Bio-Rad # 1610737EDU), boiled for 3 minutes, and spun at 21,000 x g for 1 minute. Samples were loaded onto Bio-Rad mini Protean TGX 4–20% gels (# 4561096) and run at 200V for 30 minutes. Protein was transferred at 350 mA for one hour to Immobilon PVDF membrane (Millipore-Sigma # IPVH00010). Blots were blocked for one hour in PBST (1x PBS with 0.1% Tween) with 5% nonfat milk, and stained with primary antibodies (courtesy of Maria Cristina Gambetta [27 (link)], 1:1000 in PBST with 3% BSA) for one hour. Blots were then washed 3 times for 3 minutes rotating in PBST and probed with an HRP-conjugated anti-Rabbit secondary antibody (Rockland Trueblot, # 18-8816-33, 1:1000 in PBST with 5% milk) for one hour. After extensive washing with PBST, blots were developed with Clarity ECL reagents (Bio-Rad # 1705060) and imaged. Validation of the loss of maternal dCTCF is shown in S1 Fig .
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