13 research outputs found
Performance of random forest model for cocaine and saline treatment measured by 5-fold cross validation.
<p>Performance of random forest model for cocaine and saline treatment measured by 5-fold cross validation.</p
Importance score of variables from random forest model.
<p>Importance score of variables from random forest model.</p
Summary of the link between histone modifications and alternative splicing from published studies.
<p>Summary of the link between histone modifications and alternative splicing from published studies.</p
Contribution of histone modifications to the regulation of alternative splicing.
<p>Unbiased global analysis reveals that the enrichment of specific histone marks varies with type of alternatively spliced exon. H3K36me3 and H3K4me1 show a much stronger association with alternative splicing than H3K27me3 and H3K9me2. H3K36me3 is maximally enriched at alternative PolyA exons, while at promoters it is depleted and H3K4me3 is maximally enriched.</p
Differential enrichment of HPTMs at splice junctions between alternatively spliced exon and constitutive exon.
<p><i>p</i>-value is corrected by Benjamini-Hochberg method. Significant p values are highlighted in blue [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005602#pcbi.1005602.ref023" target="_blank">23</a>].</p
Accuracies of random forest models built on different HMs and combination of HMs.
<p>Accuracies of random forest models built on different HMs and combination of HMs.</p
Mass spectrometry identifies proteins present at tagged inhibitory synapses.
<p>(A) All peptides were evaluated individually, for their presence or absence in the sample isolated via VGABA<sub>A</sub>Rα1 or eGFP, using information from peptide fragmentation spectrum (MS/MS), peptide mass spectrum (MS), and peptide retention time in extracted ion chromatogram. An example is shown for peptide, GDDNAVTGTK, from GABA<sub>A</sub>Rβ2. V: Venus. G<sub>A</sub>R: GABA<sub>A</sub> receptor. (B) Schematic representation of the cortical inhibitory synaptic protein complex. These synapses contain a multitude of inhibitory receptors, as well as cell signaling and adhesion proteins, but are entirely lacking in cell signaling molecules. The localization of LHFPL4 and Neurobeachin is hypothetical. Complete information on each peptide is in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039572#pone-0039572-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039572#pone.0039572.s002" target="_blank">Figure S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039572#pone.0039572.s003" target="_blank">Table S1</a>.</p
Biochemical purification of a tagged inhibitory synaptic protein complex.
<p>(A) Immunoblotting of various proteins shows that detergent solubilized protein extract S3 was enriched in both inhibitory (VGABA<sub>A</sub>Rα1, GABA<sub>A</sub>Rα1, GABA<sub>A</sub>Rβ2/3, GABA<sub>A</sub>Rγ2) and excitatory (GluR2, PSD95) synaptic proteins, as well as mitochondria (COx). Gel filtration of fraction S3 enabled enrichment of synaptic protein complexes relative to intracellular proteins, as shown by the specific exclusion of the endoplasmic reticulum marker BIP, from the high molecular weight fractions (6–10). Protein concentration of each fraction was measured (top), and the void volume determined by the elution of Blue Dextran (2000 kDa). Identical results were obtained for endogenous proteins in fractions prepared from wildtype or Otx1-eGFP cortices (not shown). (B) Fractions 6–10 (red box in A) from Otx1-VGABA<sub>A</sub>Rα1 or Otx1-eGFP control were pooled and subject to co-immunopurification using an anti-eGFP antibody. Immunoblotting confirmed the specific presence of inhibitory synaptic proteins (VGABA<sub>A</sub>Rα1, GABA<sub>A</sub>Rα1, GABA<sub>A</sub>Rβ2/3, GABA<sub>A</sub>Rγ2) and the absence of excitatory synaptic (GluR2, PSD95) and mitochondrial (COx) proteins in the material immunopurified via VGABA<sub>A</sub>Rα1. Only soluble eGFP was detected in the control sample. IN: Input. FT: Flow-through. IP: Immunoprecipitate. V: Venus. G<sub>A</sub>R: GABA<sub>A</sub> receptor. Further biochemical experimental results are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039572#pone.0039572.s001" target="_blank">Figure S1</a>.</p
Inhibitory synaptic proteins identified by mass spectrometry.
<p>Proteins were purified via VGABA<sub>A</sub>Rα1 or eGFP and identified by LC-MS/MS. Two replicate datasets were analyzed using the GPM Database and those proteins identified in the eGFP controls were excluded from the list. Proteins present in the dataset were confirmed to be present in the replicate by either MS or MS/MS, as indicated. GPM E value is the probability that an assignment occurs by chance. Total peptide number is the total number of peptides that match a given protein. When more than one homologue is reported, unique peptide no. corresponds to the number of peptide matches that are unique to the homologue.</p
The inhibitory synapse affinity tag, Venus-GABA<sub>A</sub>Rα1, is functional <i>in vitro</i>.
<p>(A) Schematic of VGABA<sub>A</sub>Rα1, showing the N-terminal fusion of an affinity tag, Venus. (B) Representative GABA-evoked currents (left) and current amplitude quantification (right) in voltage clamped <i>Xenopus</i> oocytes after coinjection of the indicated <i>GABR</i> cRNA subunits. Values are expressed as mean ± SEM; n  = 5 oocytes per group (**p<0.01 t-test). (C) Schematic of the patch and stimulation electrodes used for paired-pulse recordings in cultured hippocampal neurons transduced with lentivirus encoding GABA<sub>A</sub>Rα1 (Lv-G<sub>A</sub>Rα1) or Venus-GABA<sub>A</sub>Rα1 (Lv-VG<sub>A</sub>Rα1) subunits. (D) Representative traces of GABAergic transmission in paired-pulse recordings in non-infected neurons and in neurons infected with the indicated lentivirus. Control traces in the presence of bicuculine are shown below each trace. (E) Quantification of the first eIPSC amplitudes and of the paired-pulse ratios obtained in the indicated neuronal cultures. Values are expressed as mean ± SEM; n  = 7−9 recorded cells per group.</p