, 2008). For details see the Supplemental Experimental Procedures. Mouse primary cortical neurons (DIV9) were transfected with plasmids carrying scrambled or Cyfip1 shRNA and Cyfip1 WT or mutants using a calcium phosphate method ( Sans et al., 2003). At DIV14, neurons HCS assay were fixed for 20 min in PFA/SEM (4% PFA, 0.12 M sucrose, 3 mM EGTA, 2 mM MgCl2 in PBS).

Primary cortical neurons were fixed with 4% paraformaldehyde (PFA/SEM), permeabilized with 0.2% Triton X-100, and incubated overnight with the antibodies, as indicated in the Supplemental Experimental Procedures. Confocal images were obtained as described in the Supplemental Experimental Procedures. Immunoblotting was performed using standard protocols. Antibodies list and usage is described in the Supplemental Experimental Procedures. See the Supplemental

Experimental Procedures. A confocal laser-scanning microscope (Nikon) with 40× or 60× oil objectives with sequential acquisition setting at 2,048 × 2,048 pixels resolution was used. For immunofluorescence (IF), only a z series was acquired; for spine analysis, each image was a z series projection, of ∼7 to 9 images each, averaged two times and taken at 0.8 μm depth intervals. Images http://www.selleckchem.com/products/at13387.html were processed and analyzed with ImageJ 1.44 software. Five 20 μm segments starting at least 25 μm from the cell soma were analyzed for each neuron. F-EGFP or DiI staining was used to outline the profile of the dendritic shaft and protrusions. Maximal spine head width (WH), neck width (WN), and length (L) were measured for each dendritic protrusion. Spines were defined as follows: Stubby (L ≤ 1 μm), Mushroom (1 < L ≤ 3 μm; WH ≥ 2 × WN), Long Thin (1 < L ≤ 3 μm; WH < 2 × WN), and Filopodia (3 < L ≤ 5 μm). At least ten randomly chosen neurons/condition from three independent cultures were imaged for quantification. Counts and data analysis were conducted blind to experimental condition. Cortical

synaptoneurosomes were prepared as previously described (Napoli et al., 2008). Pre- and postsynaptic fractions were isolated from cortical synaptoneurosomes almost as previously described (Phillips et al., 2001). See the Supplemental Experimental Procedures for details. Primary mouse cortical neurons were prepared as previously described (Ferrari et al., 2007). See the Supplemental Experimental Procedures for details and treatments with BDNF, cycloheximide, and actinomycin D (Sigma). The procedure was slightly modified from Napoli et al. (2008). See the Supplemental Experimental Procedures for details. See the Supplemental Experimental Procedures. HEK293T cells were used as packaging cells and transfected by the calcium phosphate method (Chen and Okayama, 1987) with second generation plasmids (pLKO.1, Mission shRNA, Sigma-Aldrich) (Naldini et al., 1996) carrying scrambled or Cyfip1 shRNAs. See the Supplemental Experimental Procedures for details.