Animal use: Pregnant Sprague-Dawley rats were purchased from Harlan Laboratories (Indianapolis, IN) and kept for a few days in the local animal facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (Animal Welfare Assurance Number PHS A3269-01). The animals were handled in strict compliance with Protocol #21708(01)1B, approved by the University of Colorado Health Sciences Center Animal Care and Use Committee and with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health. For tissue collection rats were sacrificed under terminal isoflurane anesthesia, and every effort was made to minimize suffering.
Antibody specificities, hosts and sources: Golph3 and Tgoln1 (rabbit polyclonal) were generous gifts of Dr. C. Wu, then at University of Colorado School of Medicine. Kpna2 (chicken polyclonal), Man2a1 (mouse monoclonal), Mcm7 (mouse monoclonal), Pcna (rabbit polyclonal), Pdia6 (chicken polyclonal), and Uba1 (rabbit polyclonal) from Abcam (Cambridge, MA). Rpn1 (rabbit polyclonal) from Abgent (San Diego, CA). Hsp90ab1 (rabbit polyclonal) from Affinity Bioreagents, Inc. (Golden, CO). Rps3 (rabbit polyclonal) from Cell Signaling Technologies, Inc. (Beverly, MA). Psme3 (rabbit polyclonal) from Enzo Life Sciences (Farmingdale, NY). Eef1a1 (mouse monoclonal) and Psma (anti-α 1, 2, 3, 5, 6, 7; mouse monoclonal) from EMD Millipore (Billerica, MA). Cell culture supplies were from Invitrogen/Life Technologies (Carlsbad, CA). Other chemicals, unless stated otherwise, were from Sigma-Aldrich (St. Louis, MO) or Thermo Fisher Scientific (Pittsburgh, PA) and of the highest quality available.
Growth cone isolation
GCPs were prepared as described previously 
. Briefly, whole brains from gestation day-18 fetal rats were homogenized in 0.32 M sucrose containing 1 mM MgCl2
, 2 mM TES buffer (pH 7.3), and 2 µM aprotinin. Low-speed (1660×g, 15 min) supernatant (LSS) of the homogenate was layered onto a discontinuous density gradient (0.83 and 2.66 M sucrose containing MgCl2
and TES buffer) and spun to equilibrium at 242,000×g at 4°C for 40 min in a vertical rotor (VTi50, Beckman). The GCP fraction at the 0.32/0.83 M-sucrose interface and a sample of the gradient's supernatant (0.32 M sucrose) were collected.
Plating of GCPs in laminin-coated wells
24-well tissue culture plates were coated with 20 µg/ml of laminin in Hank's balanced salt solution for 3 h and rinsed three times with phosphate-buffered saline (PBS) supplemented with 0.9 mM CaCl2 and 0.5 mM MgCl2 (PBS-Ca+2/Mg2+). Wells were blocked with 5% non-fat dry milk in PBS for 30 minutes and rinsed again.
GCPs were gradually diluted with 2× modified Krebs buffer (0.05 M sucrose, 0.1 M NaCl, 5 mM KCl, 22 mM HEPES, 10 mM glucose, 1.2 mM NaH2PO4, 1.2 mM MgCl2, 2 mM CaCl2, pH 7.3) and incubated at 37°C for 10 min. After incubation equal aliquots of the GCP suspension were transferred into the 24-well culture dish, spun at 4,000 rpm for 15 min and incubated at 37°C for 30 min. Under these conditions the resealed and viable GCPs attached to the laminin-coated plastic.
Attached GCPs were extracted in two steps: (1) Brij Buffer (5 mM MgCl2
, 1 mM CaCl2
, 1% Brij 98 in PBS) was added for incubation at 37°C for 20 min, followed by 40 min at 4°C. The supernatant was collected as the Brij-soluble fraction. (2) Remaining adherent structures were rinsed 2 times with PBS-Ca+2
and lysed in 1% SDS. A chloroform/methanol (C/M) precipitation 
was performed with all samples, and the pellets were resuspended in 5% SDS and used for electrophoresis.
Controls: (a) Laminin-coated wells without GCPs. These were extracted with 1% SDS and the supernatant C/M-precipitated. The pellets were solubilized in 5% SDS. (b) Aliquot of the gradient's supernatant (clear; containing soluble proteins of the brain homogenate). This sample was C/M precipitated and solubilized in 5% SDS.
Gel electrophoresis and sample preparation for mass spectrometry
Protein samples (50–60 µg/lane), alongside with dual-colored Precision Plus Protein™ standards (Bio-Rad Laboratories, Inc., Hercules, CA), were resolved by SDS-polyacrylamide gel electrophoresis using NuPAGE Novex Bis-Tris gradient gels (Invitrogen, Carlsbad, CA). After separation, the gel was stained with Coomassie Blue (Invitrogen). Each lane of the gel was cut into 25 equal-sized pieces, which resulted in a range of 30–400 (average about 80) proteins per slice. Proteins in the gel were digested as follows. Bands were destained in 200 µl of 25 mM ammonium bicarbonate in 50% v/v acetonitrile (ACN) for 15 min and then incubated in 200 µl of 100% ACN for 15 min at room temperature. After addition of dithiothreitol (DTT) to a final concentration of 10 mM, slices were incubated at 65°C for 30 min to reduce the disulfide bonds. The reduced cysteines were alkylated with the addition of iodoacetamide (IAA) at a final concentration of 20 mM (30 min at room temperature in the dark). The IAA was removed, and washes were performed with 200 µl of distilled water followed by addition of 100 µl of ACN. ACN (i.e., all liquid) was removed in a vacuum concentrator to allow complete gel rehydration thereafter. We added 50 µl of 0.01 µg/µl trypsin in 25 mM ammonium bicarbonate (~pH 7.4) to each sample and allowed the gel pieces to rehydrate at 4°C for 30 min, before incubation at 37°C overnight. The tryptic mixtures were acidified with formic acid (FA) up to a final concentration of 1%. Peptides were extracted three times from the gel slices using 50% ACN and 1% FA, concentrated by SpeedVac to a desired volume (~18 µl), and subjected to LC-MS/MS analysis. If necessary, they were stored at −20°C.
Liquid Chromatography–Tandem Mass Spectrometry
Nano-flow reverse phase LC-MS/MS was performed using a capillary HPLC system (Agilent 1200, Palo Alto, CA) coupled via an electrospray ionization source to a linear ion trap hybrid ion cyclotron resonance mass spectrometer (LTQ-FT Ultra, ThermoFisher; San Jose, CA).
Eight microliters of the tryptic peptides were pre-concentrated and desalted on a C18 trap column ZORBAX 300SB-C18, (5 µm i.d. ×5 mm, Agilent Technologies, Santa Clara, CA) with 5% ACN, 0.1% FA at a flow rate of 15 µl/min for 5 minutes. The separation of the tryptic peptides was performed at a flow rate of 380 nl/min on a C18 reverse phase column (75 µm ID×360 µm OD×100 mm length) packed in-house with 4-µm, 100-Å pore size C18 reversed-phase stationary phase (Synergy, Phenomenex, Torrance, CA), kept at a constant 40°C using an in-house built column heater. The mobile phases consisted of 5% ACN with 0.1% FA (A) and 95% ACN with 0.1% FA (B), respectively. A 90-min linear gradient from 5 to 50% B was typically used. Data acquisition was performed using the instrument-supplied Xcalibur (version 2.0.6) software. The LC runs were monitored in positive ion mode by sequentially recording survey MS scans (m/z 400–2000) in the ion cyclotron resonance cell, while three MS/MS were obtained in the ion trap via collision-induced dissociation for the most intense ions. Once ions were selected for MS/MS they were reacquired after 30 seconds and then excluded for 90 seconds.
Database searching, protein identification
MS and MS/MS spectra were de-isotoped, centroided from raw data files and converted into “mgf” files using an in-house script. Mascot (version 2.2; Matrix Science Inc., London, UK) was used to perform database searches against rat sequences in the SwissProt database using the extracted data. Parent MS tolerance was set at ±10 ppm with MS/MS tolerance set at ±0.6 Da. Trypsin specificity was used allowing for 1 missed cleavage. The modifications of methionine oxidation, protein N-terminal acetylation, and peptide N-terminal pyroglutamic acid formation were allowed for, and cysteine carbamidomethylation was set as a fixed modification.
Scaffold version 2 (Proteome Software, Portland, OR, USA) was used to validate and compare MS/MS based peptide and protein identifications. All “.DAT” files (from Mascot) for the 25 gel bands were loaded together as one “biological sample” within Scaffold. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least two identified unique peptides. Proteins are referred to by their official encoding gene names (Entrez Gene; NCBI, U.S. National Library of Medicine). A list of abbreviations and protein names is provided in Table S1
Protein identifications and number of identifying spectra for each fraction were exported from Scaffold software. The following parameters were used: 99% confidence limit for protein identification; minimum number of peptides, 2; minimum confidence level for peptide identification, 95%. For the purposes of the present overall growth cone analysis, data for Brij-soluble and Brij-insoluble fractions were combined. “Background”, i.e., potentially contaminating polypeptides from the laminin plating matrix and the gradient supernatant, were subtracted as explained and illustrated in Results
. The net growth cone (GCP) data were used for further analysis. The raw data are shown in Table S2
For annotation analysis, gi protein accession numbers were uploaded into the DAVID (Database for Annotation, Visualization and Integrated Discovery) informatics tool (DAVID Bioinformatics Resources 6.7; 
). For GO Term (Gene Ontology) analysis we studied the “Cellular Component” and “Biological Process” categories using the GO FAT default settings. At these settings the program uses a subset of GO Terms depleted of the broadest terms (primarily from the top 5 levels of the tree) to avoid overshadowing of the more specific terms (term specificity defined on the basis of the number of child terms in the hierarchy; see DAVID website). Use of GO FAT generated more informative results than that of any specific GO Term level. For functional annotation searches we set the following parameters: “Cellular Component”, threshold count 5, EASE 0.1 (resulting in 171 chart records); “Biological Process”, threshold count 5, EASE 0.1 (resulting in 481 chart records); for functional annotation clusters
(“Biological Process”), medium stringency (resulting in 194 clusters). For KEGG pathway searches the parameters were: Threshold count 5 (resulting in 54 records).
Enrichment values (GO Terms), enrichment scores (annotation clusters), and statistical determinants (p values and Benjamini coefficients) are those calculated by DAVID software. The Benjamini coefficients are Benjamini-Hochberg corrected p values. They are adjusted for multiple comparisons to lower the family-wise false discovery rate and, thus, are more conservative than Fisher Exact p values.
Fetal brain homogenate (H), LSS and pelleted GCPs (equal amounts of protein loaded) were analyzed by SDS gel electrophoresis as described above. Resolved proteins were transferred electrophoretically onto Immobilon P (EMD Millipore Co., Billerica, MA) membranes. Membranes quenched in Tris-buffered saline with 5% non-fat evaporated milk and 0.1% Tween-20 were probed with primary and fluorescent secondary antibodies (Alexa Fluor 647, 555 or 488; Life Technologies/Invitrogen Co., Carlsbad, CA) and analyzed with a Typhoon 9400 multi-mode imager (GE Healthcare, Pittsburgh, PA). Experiments were done in triplicate. After background subtraction, densities of antigen bands (determined with Imagequant software) were normalized to H levels and values averaged. Significance of H versus GCP levels of immunoreactivity was determined using Student's t test. To assess the specificity of the other antibodies used for immunofluorescence we followed the same electrophoresis and Western blot protocols.
Immunofluorescence analysis of neurons in culture
Hippocampi from 18-day gestation Sprague-Dawley rat fetus were cut into explants or dissociated with trypsin. Explants or dissociated hippocampal pyramidal neurons were cultured on laminin-coated coverslips (Assistant brand) in B27 neurobasal medium supplemented with 10% v/v fetal bovine serum for 1–3 hours and then in serum-free B27 medium. After 1–2 days incubation at 37°C, 5% CO2 neurons were fully polarized and processed for indirect immunofluorescence.
Cultures were fixed using slow infusion of fixative [4% w/v paraformaldehyde, 0.1 M phosphate buffer (pH 7.4), 120 mM glucose, 0.4 mM CaCl2
] as developed for electron microscopic analysis 
. Cultures were rinsed (3×) with PBS containing 1 mM glycine, permeabilized with blocking buffer (PBS, 1% w/v bovine serum albumin) plus 1% (v/v) Brij™ 98 detergent for 2 min at room temperature, and washed (3×) with blocking buffer. Samples were labeled with primary antibody in blocking buffer for 1 hour at room temperature and washed 3× with blocking buffer. This process was repeated with Alexa Fluor 488-(green) or 594-(red) conjugated secondary antibodies and with red or green fluorescent phalloidin to label filamentous actin (Molecular Probes, Eugene, OR). These coverslips were mounted on slides using a polyvinyl alcohol/glycerol anti-fade medium. Samples were analyzed with a Zeiss Axiovert 200 M microscope operated by Metamorph Software (Molecular Dynamics, Sunnyvale, CA) and equipped with Zeiss lenses, epifluorescence and total internal reflection fluorescence (TIRF) optics, and a Cook Sensicam digital camera.