Donkey anti rabbit 680

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.

GFP-BAF levels in gfp-baf and gfp-baf^A13T were assessed using an SDS-PAGE and western analysis. Proteins were extracted from ovaries of less than 1-day-old females, with one ovary equivalent loaded per well. Proteins were separated by electrophoresis on a 4–20% Tris gel and blotted onto nitrocellulose membranes. Membranes were probed with primary antibodies against GFP [rabbit 1:2,000, Life Technologies], and alpha-Tubulin (mouse 1:20,000, Sigma, T5168) and fluor-conjugated secondary antibodies (donkey anti-rabbit 680, 1:20,000, LI-COR and goat anti-rabbit 800, 1:10,00 LI-COR), and detected using WesternBright Quantum kit (Advansta, K-12042-D10).