Animals and tissue dissection
All animal experiments were approved by and carried out in accordance with the guidelines from the Norwegian Committee for Experiments on Animals (“Forsøksdyrutvalget”, reference number: 20102702 and 2113555). Care was taken to ensure minimal suffering of the animals at all stages of the experiments.
Adult female outbred Sprague–Dawley and male Wistar rats (Mollegaard, Denmark), with body weight of approximately 250 g, were housed for one week before conducting the experiments. Inbred C57BL/6 mice were housed for 5, 10 or 30 days after birth (P5, P10 or P30, respectively), before sacrifice. Rats were anesthetised by isoflurane gas (Isoba vet; Schering-Plough, Denmark) and sacrificed by decapitation. Brain- and non-central nervous system (non-CNS) tissue samples for gene and protein expression analysis were dissected and immediately frozen on dry ice. Cortical tissue samples were extracted from a matrix of side-by-side areas of the adult rat neocortex, covering the occipital-, temporal- and parietal lobe (as depicted in Additional file 1). The area corresponding to the primary auditory cortex was first identified, and subsequently used as a starting point for the dissection of consecutive samples. The whole neocortex (left hemisphere) was isolated, and a total of 25 samples were extracted. Each tissue sample measured approximately 2x2 mm and was dissected from corresponding neocortical areas from six individual rats. All tissue samples were stored at -80°C. For in situ RNA hybridisation and immunohistochemistry analysis, rats and mice were first anesthetised by isoflurane gas, followed by intraperitoneal (i.p.) injection of pentobarbital and transcardiac perfusion with 9 mg/ml NaCl and 4% (w/v) paraformaldehyde/PBS. Fixated brains were placed in PBS, soaked in 30% (w/v) sucrose and embedded in Tissue-Tech O.C.T. compound (Sakura Finetek, USA). The embedded brains were frozen on dry ice and stored at -80°C. For pre-embedding electron microscopic immunocytochemistry, rats were anesthetised with pentobarbital (100 mg/kg, i.p.) before fixation through transcardiac perfusion with a solution of 4% formaldehyde in 0.1 M sodium phosphate buffer, pH 7.4 (PB) (50 ml/min for 15 min). The fixed brains were stored in the fixative diluted 1:10 in PB at 4°C.
RNA purification, cDNA synthesis and gene expression analysis
The tissue samples from rat were homogenised using a Beadmill TissueLyser (Qiagen, Germany), and total RNA was purified from homogenised samples using the ABI PRISM™ 6100 Nucleic Acid PrepStation (Applied Biosystems, USA). The NanoDrop® ND-1000 spectrophotometer (Nanodrop Technologies, USA) was used to measure the RNA quantity and quality. 20 ng total RNA from each sample was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Total RNA from human brain tissues (i.e. fetal and adult brain, frontal-, temporal- and occipital pole, hippocampus, medulla and cerebellum) was obtained from Clontech (USA). Quantitative real-time PCR (qRT-PCR) was conducted using the ABI Prism 7900HT sequence detector system (Applied Biosystems). The samples were run in triplicates, as previously described , and the comparative Ct method  was used to determine the relative gene expression levels. The expression level of hypothetical protein LOC689986 (LOC689986, [GenBank: NM_001109563.1]) and the human orthologous gene Chromosome 1 open reading frame 146 (C1orf146, [GenBank: NM_001012425.1]) was measured using TaqMan® Assay probes (Product id: Rn01765992_m1 and Hs00415950_m1, rat and human, respectively) (Applied Biosystems). The expression levels were normalised relative to the endogenous controls acidic ribosomal phosphoprotein P0 (Arbp) and/or β-actin (Actb).
In addition, the Tissue Gene Expression Database (Human Body Map, Applied Biosystems), consisting of 32 different human tissue samples (http://www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GSE7905), was mined in order to screen for expression of the human orthologous gene.
Cloning and generation of eukaryotic expression vectors
cDNA generated from an adult rat temporal cortex sample was used as template to amplify the full length LOC689986 transcript; forward primer sequence: 5′-ACAGCCACCCACCCCACA, reverse primer sequence: 5′-GTGTTCCTCTGCAGGAATAGC. The amplified gene was cloned into the pCR®II-TOPO® vector (Invitrogen, USA). To generate a vector encoding C-terminally V5-tagged LOC689986, the gene was amplified from the above described vector and ligated into the pcDNA™3.1⁄V5-His A vector (Invitrogen) via its BamHI/ApaI sites. To generate vectors encoding C- or N-terminally YFP, the gene was amplified from the pCR®II-TOPO® vector and ligated into the pEYFP-C1 or pEYFP-N1 vector (Clontech, USA) via its EcoRI/BamHI or NheI/BamHI sites, respectively.
Probe preparation and in situ RNA hybridisation
Antisense and sense riboprobes were generated by T7 and SP6 transcription from linearised plasmid (LOC689986 gene cloned into the pCR®II-TOPO® vector) in the presence of digoxigenin labelling mix (Roche, Switzerland). 30 μm thick coronal cryosections were cut through the whole adult rat brain, using a Leica CM3050 cryostat, and floating sections were treated as previously described . In short, sections were permeabilised with Proteinase K (20 μg/ml), fixated in 4% (w/v) paraformaldehyde/PBS, treated with 25% (v/v) acetic anhydride in 0.1 M TEA (pH 8), following application of riboprobes in hybridisation buffer to the sections. Sense riboprobes were included in all experiments as a negative control. The hybridisation reaction was left for at least 16 hours at 60°C, and the sections were then washed thoroughly prior to RNase A treatment (20 μg/ml). Alkaline coupled anti-digoxigenin antibody was applied (diluted 1:2000) and visualisation was achieved by using NBT/BCIP chromogen substrates (Roche).
Production of rabbit anti-LOC689986 peptide antibody
A polyclonal peptide antibody, targeting a C-terminal epitope with amino acid sequence: IEQSPVWRTLQK, was generated in rabbits by 21st Century Biochemicals (Marlboro, MA, USA). Polyclonal serum was affinity purified and the peptide antibody was subsequently used in western blot- and immunohistochemistry analysis.
Protein determination, gel electrophoresis and western blot analysis
Homogenised tissue samples from rat and cell lysates from transiently transfected HeLa cells were prepared in RIPA Triton X-100 buffer (150 mM NaCl, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% SDS (w/v) and 50 mM Tris/HCl pH 8.0). Protein concentrations were determined using the DC Protein Assay Kit (Bio-Rad, USA). Polyacrylamide gel electrophoresis and immunoblotting were performed according to the manufacturer’s instructions using NuPAGE®Bis-Tris pre-cast gels 10% (Invitrogen). Primary antibodies used were: rabbit anti-LOC689986 peptide antibody (21st Century Biochemicals), mouse anti-V5 (Invitrogen), goat anti-Gapdh and goat anti-Actin (Santa Cruz Biotechnology). Secondary antibodies used were: donkey anti-mouse IgG-HRP, donkey anti-rabbit IgG-HRP and donkey anti-goat IgG-HRP (Santa Cruz Biotechnology). Enhanced chemiluminescence (GE Healthcare, United Kingdom) was used for detection, and equal protein loading was examined by either Gapdh or Actin immunodetection. Pre-absorption controls were included by incubating the anti-LOC689986 antibody with the peptide used to generate the antibody (1 hour at room temperature) prior to use.
20 μm sagittal cryosections were cut from embedded mouse brains using a Leica CM3050 cryostat, collected and thaw-mounted onto SuperFrost
Plus slides (Thermo Fisher Scientific, USA). Sections were dried for 30 min at 37°C and rinsed briefly in PBS. After blocking in 5% (w/v) bovine serum albumin (Sigma-Aldrich, USA) and 0.2% (v/v) Triton X-100 in antibody buffer (150 mM NaCl, 50 mM Tris–HCl, 1% (w/v) bovine serum albumin, 100 mM L-Lysine, 0.04% (w/v) Sodium Azide) for 1 hour at room temperature, primary antibodies were applied and the slides were incubated at 4°C overnight. Primary antibodies used were: rabbit anti-LOC689986 peptide antibody (diluted 1:200, 21st Century Biochemicals) and mouse anti-200 kD Neurofilament Heavy Monoclonal antibody (diluted 1:500, Abcam, United Kingdom). Slides were washed three times in PBS, and incubated for 2 hours at room temperature in highly cross-absorbed fluorescent-conjugated secondary antibodies (Invitrogen); Alexa Fluor® 488 goat anti-rabbit IgG (diluted 1:1000) and Alexa Fluor® 594 goat anti-mouse IgG (diluted 1:1000). Nuclei were stained using DAPI. Slides were mounted using Vectashield mounting medium (Vector Labs, USA) and fluorescent images were obtained by a Zeiss LSM 510 META (Zeiss, Germany) or Leica TCS SP2 AOBS (Leica Microsystems, Germany) confocal microscope.
Cell culturing, transient transfection and immunocytochemistry
Human HeLa cells (ATCC-LGC, USA) were cultivated in Eagle’s Minimum Essential Medium supplemented with 10% (v/v) fetal bovine serum and penicillin/streptomycin. Cells were transiently transfected for 24–48 hours using Lipofectamine 2000 Transfection Reagent (Invitrogen) according to the recommendations of the manufacturer. Transiently transfected cells were grown on cover slips and fixated using 4% (w/v) paraformaldehyde/PBS for 45 min. Cells were permeabilised for 15 min by subjecting them to 0.5% (v/v) Triton X-100 in PBS treatment. Detection of recombinant protein was achieved either directly (transient transfection using vectors encoding either a C- or N-terminal YFP tagged recombinant protein), or by using mouse anti-V5 primary antibody (diluted 1:1000, Invitrogen) and Alexa Fluor® 594 goat anti-mouse IgG (diluted 1:1000, Invitrogen) secondary antibody. Nuclei were stained with DAPI. Images were obtained by using a Leica TCS SP2 AOBS confocal microscope (Leica Microsystems).
Pre-embedding electron microscopic immunocytochemistry
Frontal sections (50 μm) of two fixed rat brains were cut on a vibratome, and labelled free-floating with the rabbit anti-LOC689986 peptide antibody (diluted 1:50, 21st Century Biochemicals) according to a three-layer immunoperoxidase method, in which the antigen-antibody binding is visualised by an electron dense diaminobenzidine reaction product. To preserve the ultrastructural morphology, the sections were processed without detergent. Samples containing layer 1–3 of somatosensory cortices were dissected out of the stained sections, dehydrated and embedded in Durcupan ACM Fluka (Sigma-Aldrich). Then ultrathin sections (gold colour) were cut on 300 mesh nickel grids. The ultrathin sections were viewed in a Tecnai 12 electron microscope and electron micrographs at x43,000 magnification were taken in layer 2 at both surfaces of the sections.
Protein-protein interaction analysis
A yeast-2-hybrid (Y2H) screen was performed by using the full LOC689986 open reading frame as bait to screen both adult and embryonic (E10.5-E12.5) mouse brain libraries. The analysis was performed by using the ULTImate Y2H™ screen at Hybrigenics Services (France, http://www.hybrigenics-services.com/). A total of 88.47 and 65.1 million interactions were analysed in the embryonic and adult mouse brain libraries, respectively. Hybrigenics assigns a statistical confidence score, the Predicted Biological Score (PBS®), to each interaction. In short, interacting proteins are ranked according to both local and global technical parameters to compute the final score. The PBS® is computed as an expected value (e-value), ranging from 0 (specific interaction) to 1 (probable artefact). For practical purposes these scores are divided into four categories, ranging from A (close to 0, very high confidence in the interaction) to D (close to 1, very low confidence in the interaction) . More details regarding the scoring and ranking of the protein-protein interactions can be found at the Hybrigenics homepage (http://www.hybrigenics-services.com/contents/our-services/discover/ultimate-y2h-2/ultimate-deliverables).
Web-based bioinformatic tools
Genomic searches were performed using the UCSC Genome Bioinformatics database and the NCBI database. BlastView from the Ensembl Genome Browser (release 63)  was used to search for homologous sequences in the rat genome database. BlastView was also applied to search for orthologous sequences in both vertebrate and invertebrate species (i.e. Caenorhabditis elegans and Drosophila melanogaster), as well as a yeast genome database (i.e. Saccharomyces cerevisiae). All searches were conducted using BLAT default settings. Nucleotide sequences were retrieved from the NCBI and UCSC databases. Multiple sequence alignments were performed using ClustalW2 from EMBL-EBI applying default settings (http://www.ebi.ac.uk/Tools/services/web/toolform.ebi?tool=clustalw2) [19, 20]. The sequence conservation between various vertebrate species was analysed by exploring the UCSC database. Genetic synteny analysis was performed by exploring the Genomicus v64.01 database, using default settings (http://www.dyogen.ens.fr/genomicus-64.01/cgi-bin/search.pl). In order to examine whether LOC689986 belongs to known protein families or contains known domains, regions or sites, InterProScan Sequence Search from EMBL-EBI was used (http://www.ebi.ac.uk/Tools/pfa/iprscan/) . Prediction of signal peptide cleavage sites was performed by the SignalP 3.0 Server from the Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/SignalP/) [22–24]. MyHits was explored to examine potential motifs and post translational modifications of the predicted protein (http://myhits.isb-sib.ch/cgi-bin/motif_scan). Finally, we used the PSIPRED Protein Structure Prediction Server from the UCL-CS Bioinformatics (http://bioinf.cs.ucl.ac.uk/threader/), to analyse the predicted LOC689986 amino acid sequence.