Ecl lightening plus

Tissue was lysed using RIPA buffer (50mM Tris, pH 7.4, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate) supplemented with protease inhibitors (Calbiochem, Billerica, MA). Protein samples were separated on 4–15% gradient TGX precast gels (Bio-Rad, Hercules, CA), transferred to nitrocellulose membrane (Whattman, Pittsburgh, PA). Membranes were blocked and antibodies were diluted in 5% milk in TBST (50mM Tris, pH 7.6, 150mM NaCl, 0.1% Tween 20) and analyzed using ECL Lightening Plus (Perkin–Elmer, Waltham, MA).

HeLa cells were incubated on ice with PBS (Life Technologies) for 5 min, harvested with a cell scraper (Corning) and lysed on ice in lysis buffer (1% IGEPAL630 (Sigma and Thermo Fisher), 50mM Tris pH7.4 (Life Technologies), 150mM NaCl (Ambion) in PBS) supplemented with protease and phosphatase inhibitor cocktail III (Sigma). For each immunoprecipitation 500μg of total lysate was incubated with 1–2μg of antibody for 2 h and then incubated with magnetic protein G-sepharose (GE Healthcare Life Sciences) for an additional 1.5 hours. Immunoprecipitating proteins were boiled in 2X Laemmli sample buffer with β-mercaptoethanol (Bio-Rad) or collected in pH2.0 Glycine (Life Technologies) and quenched in Tris pH9.0 (Ambion) for mass spectrometry analysis. Protein from flash frozen gestational week 23 brains were extracted using the NE-PER Kit (Thermo Fisher) followed by homogenization with a pellet pestle (Kimble). Reduced samples were separated on 4–15% TGX gels (Bio-Rad), transferred to supported BA85 Protran (GE Healthcare) and subjected to immunoblot analysis using ECL lightening Plus (Perkin-Elmer) or LiCOR Odyssey scanner for quantitative analysis.