Lowry assay method

To assess protein levels, neocortical lysates from pooled (n = 2) dorsal telencephalon of WT and Fmr1 KO embryos at E15.5 were prepared using cell lysis buffer containing 20 mM HEPES pH 7.5, 20% glycerol, 400 mM NaCl, 1 mM MgCl₂, 0.5 M DTT, 0.5 mM PMSF, 0.1% NP40, 1 × protease and phosphatase inhibitor, and 1 mM EDTA pH 8.0. Following the manufacturer’s protocol, protein concentration was measured by the Lowry Assay Method (Bio-Rad). The neocortical lysates (25 μg) were subjected to SDS/PAGE (7.5% TGX™ FastCast™ Acrylamide Kit; Bio-Rad) and transferred onto polyvinylidene difluoride membranes (Millipore) with 40 V at 4 °C for 4 h. The membranes were then blocked in 10% TBS blocking buffer (Licor) for 1 h, and incubated with a primary antibody (as described above, Immunohistochemistry). The membrane was washed with TBST (containing 0.1% Tween 20) for 1 min with three repeats and 5 min with three repeats and incubated with a secondary antibody, either donkey anti-rabbit 680 (1:10,000; Licor), or donkey anti-mouse 680 (1:20,000; Licor), diluted in 10% TBS blocking buffer for 1 h at RT under a shaded condition. The signal was detected using the ODYSSEY infrared imaging system (Licor) and quantified using ImageJ 1.48v software (National Institute of Health) with Gapdh as normalizer.

Since BRCA1 protein is predominantly expressed in the nucleus, nuclear protein extraction was performed. Cells were lysed with RSB buffer (Tris 10 mM pH 7.5, NaCl 10 mM and MgCl2 3 mM,) containing protease inhibitor (Roche). Cell pellets were then dissolved in NB buffer (Tris 10 mM pH 7.5, NaCl 0.4 mM and EDTA 1 mM) containing protease inhibitor and supernatant was collected. Protein concentration was measured by Lowry assay method (Bio-Rad) and equal amounts of protein (50μg) were separated by SDS-PAGE on 6% home-made gels. Proteins were then electrotransferred to nitrocellulose membrane (Whatman), before blocking and incubation with antibodies. Antibodies used in this study were mouse anti-BRCA1 (OP92, Calbiochem) at 1:100 dilution, mouse anti-HSP70/HSC70 (ADI-SPA-820, Enzo Life Sciences) at 1:2000 dilution (loading control) and the corresponding horseradish peroxidase (HRP) conjugated secondary antibody (Dako, Glostrup, Denmark) at 1:10000 dilution. Antibody visualization was carried out with Amersham ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences). BRCA1 protein content was determined relative to HSP70/HSC70 protein content.