2.4. Western Blotting

E12 forebrain and E12 hypothalamic tissues were harvested and Dounce homogenized in RIPA buffer (Thermo Fisher Scientific) supplemented with a Halt protease inhibitor cocktail (Thermo Fisher Scientific) and phenylmethanesulfonyl fluoride (Sigma Aldrich). Samples were sonicated briefly and centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant was then diluted in an equal volume of Laemmli’s sample buffer (BioRad), boiled for 5 min, and electrophoresed on 12% acrylamide gels. Proteins were transferred to a nitrocellulose membrane and blocked in TBS containing 0.05% Tween-20 (TBST) and 5% milk. Anti-InsR β monoclonal antibody (BD Biosciences; 610109) was applied overnight in TBST at 4°C followed by three 5 min washes in TBST. Membranes were then incubated in IRDye 800 (LI-COR Biosciences) in TBST for 1 hour at RT, rinsed in TBST, and imaged using the Odyssey Clx Infrared Imaging System (LI-COR Biosciences).