Ab17963

E10.5 embryos were dissected free of the amniotic sac, frozen in liquid nitrogen, thawed, and disrupted by pipetting in Hank’s Balanced Salt Solution containing 5 mM ethylenediaminetetraacetic acid (EDTA). Total cell lysates were prepared by heating samples in boiling Laemli buffer (66 mM Tris–HCl, pH 6.8, 2% (w/v) SDS, 10 mM EDTA). The samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis using anti-neurofibromin antibody ab17963 (Abcam). Immunoreactive bands were visualized by chemiluminescence. Quantification of individual band intensities was performed using ImageJ. One-way analysis of variance (ANOVA) was used to assess statistical differences between band intensities. Significant ANOVA results were analyzed post hoc by the Tukey-Kramer multiple comparisons test.

Total proteins were extracted in RIPA lysis buffer (Sigma) containing 1% protease inhibitor cocktail (Sigma) and 10% phosphatase inhibitor (Roche). Proteins were quantified using the Pierce BCA Protein assay kit (Thermo Fisher Scientific) and loaded using NuPAGE™ 3%–8% and Tris-acetate (Invitrogen). Total proteins were then transferred to PVDF membrane (Bio-Rad) with a Trans-Blot Turbo Transfert system (Bio-Rad), blocked with Odyssey blocking buffer (OBB) containing 0.1% Tween-20, and incubated overnight with the primary anti-neurofibromin antibody (abcam, ab17963, 1/1000) diluted in the OBB containing 0.1% Tween-20. The membrane was then incubated for 1 h with the corresponding IRDye secondary antibodies (LI-COR). Immunoreactive protein bands were revealed using an Odyssey CLx Imager (LI-COR) according to the manufacturer’s protocol. Anti-beta-Actin antibody (Santa Cruz Biotechnologies, sc4778, 1/10,000) or anti-vinculin (abcam, ab129002, 1/1000) was used to verify equal protein loading.

Immunohistochemistry was performed as described previously [74 (link), 75 (link)]. The sections were incubated with primary antibodies diluted with 3% BSA/TBST (containing 0.1% Triton X100), including goat anti-FMRP (1:1000; LS-B3953; LifeSpan Biosciences Inc.), rabbit anti-mTOR (1:1000; 7C10; Cell Signaling Technology), rabbit anti-phospho-mTOR (Ser2448) (1:1000; 2971; Cell Signaling Technology), mouse anti-Ctnnb1 (1:2000; 610153; BD Biosciences), and rabbit anti-Nf1 (1:1000; ab17963; Abcam) overnight at 4 °C. The secondary antibodies used were Cy3-conjugated donkey anti-goat IgG (1:500; Life Technologies), Cy3-conjugated donkey anti-rabbit IgG (1:500; Life Technologies), and Alexa 488-conjugated donkey anti-mouse IgG (1:500; Life Technologies), and counterstained with 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI)/TBST (1:1000; Sigma). Images were visualized by a confocal laser microscope Zeiss LSM800 (Carl Zeiss).