Journal:

Article Title: Comparative analysis of the gene-dense ACHE/TFR2 region on human chromosome 7q22 with the orthologous region on mouse chromosome 5

doi:

Figure Lengend Snippet: Mouse and human genes in the ACHE/TFR2 region

Article Snippet: Partial mouse cDNAs for Asr2 , Cip1 and Perq1 were amplified from mouse brain cDNA (Clontech).

Techniques: Variant Assay, Binding Assay, Expressing, Sequencing, Clone Assay, Functional Assay, Activity Assay, Transgenic Assay, Produced

Journal: The Journal of Biological Chemistry

Article Title: Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker *

doi: 10.1074/jbc.M111.304881

Figure Lengend Snippet: Moesin domain organization, conformational states, and binding sites. Correspondence between regions of sequence and the domain architecture of full-length moesin. Colors correspond to distinct functional parts: lobe A of FERM domain (green), lobe B (orange), lobe C (yellow), α-helical region (blue), FLAP (red), and C-terminal tail (gray). A, domains of moesin within the primary sequence. Residue numbers in human moesin are shown for boundaries and for the C-terminal phosphorylation site. B, schematic of full-length inactive and active conformations. Also shown in light blue and labeled in the figure are four known binding sites of the ERM protein that include two for PIP2 (1 and 2) and two for protein ligands (3 and 4). The two PIP-2 binding sites are shaded dark green. The two protein-binding sites are enclosed with dashed regions. a, schematic of inactive conformation (based on structure 2I1K) in which three binding sites are masked (by α-helical region, FLAP, and tail) but the PIP2-binding lysine PATCH (1) remains exposed. (The connecting peptide between the FLAP and C-terminal tail is poorly visible because it runs behind lobe C in this perspective.) b, schematic of active conformation in which three of the binding sites are unmasked. The helical region is shown with dashes in the middle to indicate it is longer than shown.

Article Snippet: POPC (PC; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine), phosphatidylethanolamine (PE; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPS (PS; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoserine), l -α-phosphatidylinositol natural from bovine liver, PI(4,5)P 2 (PIP2; l -α-phosphatidylinositol 4,5-bisphosphate) natural from porcine brain, and sphingomyelin natural from brain were all purchased from Avanti Polar Lipids.

Techniques: Binding Assay, Sequencing, Functional Assay, Labeling, Protein Binding

Journal: The Journal of Biological Chemistry

Article Title: Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker *

doi: 10.1074/jbc.M111.304881

Figure Lengend Snippet: FLAP, sequence and structure analysis. A, electrostatic surface potential of full-length insect ERM protein (PDB 2I1K) and B, of unmasked moesin FERM domain with color charge scale on the left. The molecules are oriented to show the PIP2 binding POCKET, which is covered by the FLAP in the closed conformation of moesin (A) and uncovered in the FERM domain (B). Labels indicate locations of the A, B, and C lobes. Location of PIP2 binding POCKET is indicated by arrows. C, ribbon representation of the closed ERM structure. FLAP is red (N-terminal and C-terminal parts) or magenta (reconstructed tip, amino acids 473–485). β1 to β4 strands of the PH-like domain are green; β5-β7 strands are yellow; α-helix is blue. C-terminal tail is light blue. Light Blue circles indicate locations of the two critical residues, Lys-63 and Lys-278, in the POCKET, which are masked by the FLAP when moesin is closed. D, multiple sequence analysis of the FLAP of human ezrin, radixin, and moesin and flanking regions. Red dashed rectangle highlights the FLAP. Above the sequence is a row of symbols that scores the extent of sequence conservation scored by ClustalX (* = identity; : = all residues belong to a strong conservation group; · = all residues belong to a weak conservation group). Arrowheads indicate residues in moesin that are acidic (red) or short side chains (black). Statistical significance compared with Moesin WT membrane enrichment is shown as: *, p < 0.1 and **, p < 0.01.

Article Snippet: POPC (PC; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine), phosphatidylethanolamine (PE; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPS (PS; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoserine), l -α-phosphatidylinositol natural from bovine liver, PI(4,5)P 2 (PIP2; l -α-phosphatidylinositol 4,5-bisphosphate) natural from porcine brain, and sphingomyelin natural from brain were all purchased from Avanti Polar Lipids.

Techniques: Sequencing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker *

doi: 10.1074/jbc.M111.304881

Figure Lengend Snippet: The PIP2 binding POCKET defined by crystallography is necessary for ERM activation. A, the complex of radixin FERM domain with IP3 (PDB 1GC6) represented as ribbon drawing. The dashed box highlights the basic cleft between lobes A and C. The IP3 molecule is shown in a ball and stick model. The positively charged residues proposed to interact with the phosphate groups of IP3 are shown in space-filled and colored blue. The side chains of IP3 are shown in ball-and-stick models. Inset shows expanded view of this cleft. B, membrane enrichment of GFP-tagged constructs of WT or mutant moesin (and GFP-only as control) was assessed in representative midplane confocal images of transfected Jurkat cells. The large nucleus of Jurkat is indicated with “N.” Scale bar is 5 μm. C, quantitation of membrane enrichment of moesin constructs in Jurkat cells having the indicated mutations of lysines in the PIP2-binding POCKET. The enrichment characteristic of the WT protein is indicated by the upper dashed line. Proteins not enriched at the plasma membrane typically have ratios of membrane/cytoplasmic localization of ∼1.0 (lower dashed line). Statistical significance compared with Moesin WT membrane enrichment is shown as: *, p < 0.1; **, p < 0.01; ***, p < 0.001.

Article Snippet: POPC (PC; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine), phosphatidylethanolamine (PE; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPS (PS; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoserine), l -α-phosphatidylinositol natural from bovine liver, PI(4,5)P 2 (PIP2; l -α-phosphatidylinositol 4,5-bisphosphate) natural from porcine brain, and sphingomyelin natural from brain were all purchased from Avanti Polar Lipids.

Techniques: Binding Assay, Activation Assay, Construct, Mutagenesis, Transfection, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker *

doi: 10.1074/jbc.M111.304881

Figure Lengend Snippet: Mutation of the PIP2 POCKET abolishes PIP2 binding. A, binding of WT and K63N/K278N moesin to PIP2 in LUV was measured by a cosedimentation assay over the indicated range of PIP2 concentrations. Dissociation constants (mean of two independent experiments ± S.D.) are presented in the lower panel. B and C, binding of moesin to the cytoplasmic tail of CD44 (B) and the tail of NHERF1 (C) were assessed by pulldown assays. Recombinant proteins WT and K63/278N moesin were tested in the presence of lipid control PS (phosphatidylserine) (upper panels) or PIP2 (lower panels). Regions shown are from the pellet using anti-His to detect moesin pulled down and Coomassie stain to validate equal loading of GST-CD44 or GST-NHERF1.

Article Snippet: POPC (PC; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine), phosphatidylethanolamine (PE; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPS (PS; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoserine), l -α-phosphatidylinositol natural from bovine liver, PI(4,5)P 2 (PIP2; l -α-phosphatidylinositol 4,5-bisphosphate) natural from porcine brain, and sphingomyelin natural from brain were all purchased from Avanti Polar Lipids.

Techniques: Mutagenesis, Binding Assay, Recombinant, Staining

Journal: The Journal of Biological Chemistry

Article Title: Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker *

doi: 10.1074/jbc.M111.304881

Figure Lengend Snippet: Binding of PATCH mutant constructs to PIP2. A, binding of WT and mutant moesin to PIP2 in LUV was measured by a cosedimentation assay over the indicated range of PIP2 concentrations. Moesin constructs tested included WT, each of the single lysine mutations (K253N, K254N, K262N, K263N), and double mutations of the two pairs (K253N/K254N and K262N/K263N) that strongly impaired membrane localization in cells and the combined mutation of the 4 lysines K253N/K254N/K262N/K263N (4N). B, tabulation of dissociation constant (mean of two independent experiments ± S.D.).

Article Snippet: POPC (PC; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine), phosphatidylethanolamine (PE; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPS (PS; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoserine), l -α-phosphatidylinositol natural from bovine liver, PI(4,5)P 2 (PIP2; l -α-phosphatidylinositol 4,5-bisphosphate) natural from porcine brain, and sphingomyelin natural from brain were all purchased from Avanti Polar Lipids.

Techniques: Binding Assay, Mutagenesis, Construct

Journal: The Journal of Biological Chemistry

Article Title: Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker *

doi: 10.1074/jbc.M111.304881

Figure Lengend Snippet: Single lysine mutations in the PATCH impair PIP2-induced activation of moesin binding to CD44 and NHERF1. A, binding of moesin to CD44 was assessed using pulldown of moesin-His by CD44 tail-GST immobilized on beads. Moesin constructs tested included WT, each of the single lysine mutations that strongly impaired membrane binding in cells, and combined mutations of lysines in a pair. Gel regions shown are from the pellet using anti-His to detect moesin pulled down and Coomassie stain to validate equal loading of GST-CD44. B and C, similar analysis as A but using NHERF1 tail-GST (B) or NHERF1 full-length-GST (C) instead of CD44-GST.

Article Snippet: POPC (PC; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine), phosphatidylethanolamine (PE; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPS (PS; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoserine), l -α-phosphatidylinositol natural from bovine liver, PI(4,5)P 2 (PIP2; l -α-phosphatidylinositol 4,5-bisphosphate) natural from porcine brain, and sphingomyelin natural from brain were all purchased from Avanti Polar Lipids.

Techniques: Activation Assay, Binding Assay, Construct, Staining

Journal: The Journal of Biological Chemistry

Article Title: Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker *

doi: 10.1074/jbc.M111.304881

Figure Lengend Snippet: FLAP removal increases PIP2-induced protein ligands binding to moesin. A, SDS-PAGE analysis of the pellet from pulldown assays using soluble moesin-His and the cytoplasmic tail of CD44-GST (or NHERF1-GST) immobilized on beads. Moesin in the pellet is detected by anti-His antibody. CD44-GST (or NHERF1-GST) is detected by Coomassie Blue to verify equal loading. B, quantitation of the relative amounts of bound moesin determined by densitometry.

Article Snippet: POPC (PC; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine), phosphatidylethanolamine (PE; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPS (PS; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoserine), l -α-phosphatidylinositol natural from bovine liver, PI(4,5)P 2 (PIP2; l -α-phosphatidylinositol 4,5-bisphosphate) natural from porcine brain, and sphingomyelin natural from brain were all purchased from Avanti Polar Lipids.

Techniques: Binding Assay, SDS Page, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker *

doi: 10.1074/jbc.M111.304881

Figure Lengend Snippet: Analysis of stoichiometry and cooperativity of moesin binding to PIP2. A, cooperativity. Binding of moesin-Alexa 488 to LUVs composed of varying percentages (0–15%) of PIP2 assessed by co-sedimentation assays followed by spectrofluorimetric analysis. LUVs having fixed total lipid concentration (0.28 mm accessible lipid) but vary in their mole fractions of PIP2 were added to a fixed concentration of moesin-Alexa 488 (0.4 μm). The percent of moesin bound is plotted as a function of the percentage of PIP2 in the LUVs where each point is the average of two experiments. The solid line is the nonlinear least squares best fit of all the data. B, stoichiometry. A standard protein to membrane FRET assay was employed to quantitate membrane-bound moesin. The FRET measured occurs between intrinsic tryptophan donors in moesin and the dansyl-PE acceptors in the LUVs. LUVs containing 10 mol % PIP2 in a lipid mixture mimicking the plasma membrane inner leaflet (PE/PC/PS/l-α-phosphatidylinositol/sphingomyelin/cholesterol/dansyl-PE/PIP2 (23.8:9.1:18.1:4.5:4.5:25:5:10)) were titrated into a fixed concentration of nonlabeled moesin (3.6 μm). These conditions were chosen to drive high affinity PIP2 binding such that the titration yields a linear increase in membrane-associated moesin until all proteins had been bound and a plateau achieved. High variability (a time dependent signal loss we ascribe to vesicle aggregation) was observed when LUV concentrations exceeded 6.0 μm PIP2 (data point shown in parentheses). The intersection of the best-fit straight lines for the linear increase and plateau regions represent the saturation point, yielding 3.1 ± 0.25 μm PIP2 molecules per 3.6 μm moesin molecules equivalent to a PIP2/moesin ratio of about 0.86 ± 0.07 (average of 5 experiments ± S.E.).

Article Snippet: POPC (PC; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine), phosphatidylethanolamine (PE; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPS (PS; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoserine), l -α-phosphatidylinositol natural from bovine liver, PI(4,5)P 2 (PIP2; l -α-phosphatidylinositol 4,5-bisphosphate) natural from porcine brain, and sphingomyelin natural from brain were all purchased from Avanti Polar Lipids.

Techniques: Binding Assay, Sedimentation, Concentration Assay, Titration

Journal: The Journal of Biological Chemistry

Article Title: Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker *

doi: 10.1074/jbc.M111.304881

Figure Lengend Snippet: Representation of a proposed additional mechanism for conformational change when PIP2 binds to the PATCH. Panel A shows a model of the PATCH of intact ERM interacting with a lipid bilayer including a PIP2 molecule. Lobe C, the PH-like domain, has been colored to facilitate visualization: β1–β4 strands in green, β5–β7 strands in yellow, and the α-helix in blue. The FLAP is colored red. The headgroup of PIP2 is accommodated between the two pairs of lysines (Lys-253/Lys-254 and Lys-262/Lys-263) whose side chains are shown in ball and stick representation. Panel B is a magnified view of the binding site.

Article Snippet: POPC (PC; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine), phosphatidylethanolamine (PE; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPS (PS; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoserine), l -α-phosphatidylinositol natural from bovine liver, PI(4,5)P 2 (PIP2; l -α-phosphatidylinositol 4,5-bisphosphate) natural from porcine brain, and sphingomyelin natural from brain were all purchased from Avanti Polar Lipids.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: C-terminal splice variants of P/Q-type Ca 2+ channel Ca V 2.1 α 1 subunits are differentially regulated by Rab3-interacting molecule proteins

doi: 10.1074/jbc.M117.778829

Figure Lengend Snippet: Characterization of splice variants of the CaV2.1 C terminus in the human cerebellum. A, schematic topology of VDCC CaV2.1 α1 subunit and β subunit and domain structures of CaV2.1 CTD splice variants. PolyQ is 11 glutamine (Q) repeats in the cDNA construct subjected to functional characterization. GenBankTM accession numbers are as follows: U79666 for CaV2.1 CTD (+44,47) and NM_001127221 for CaV2.1 CTD (+44,Δ47). The exon 44-coding region (GenBankTM accession number Z80152) is eliminated in CaV2.1 CTD (−44,47) and CaV2.1 CTD (−44,Δ47). The GGCAG sequence at the 5′-end of exon 47 is eliminated in CaV2.1 CTD (+44,Δ47) and CaV2.1 CTD (−44,Δ47). B, line represents the structure of the region of the human cacna1a gene containing exons 40–47 (top), exons 42–45 (bottom left), and exons 46 and 47 (bottom right). Constitutive exon is shown as black boxes, and alternative exon is shown as gray boxes. The alternatively spliced sequence GGCAG is indicated by dark gray boxes. Stop codons are indicated by asterisks. Alternative splicing patterns are indicated by lines connecting the exons. C, PCR products from human cerebellum cDNA library.

Article Snippet: Human VDCC β 4 subunit (GenBank TM accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_001005747","term_id":"1052790476","term_text":"NM_001005747"}} NM_001005747 ) was cloned using PCR from human whole-brain Marathon-Ready cDNA (Clontech) and was subcloned into pcDNA TM 3.1 (−) vector (Thermo Fisher Scientific).

Techniques: Construct, Functional Assay, Sequencing, cDNA Library Assay

Journal: Haematologica

Article Title: ALK-positive large B-cell lymphomas with cryptic SEC31A-ALK and NPM1-ALK fusions

doi: 10.3324/haematol.2009.014761

Figure Lengend Snippet: Molecular and functional analysis of the SEC31A-ALK and NPM1-ALK fusion. (A) Case 1: RT-PCR with primers specific for the 5′ end (upper figure) and 3′ end (lower figure) of ALK showed no expression on cDNA level of ALK in reactive lymph nodes, but presence of full length ALK (5′ and 3′ end) in normal brain cDNA. In case 1, only the 3′ end of ALK, containing the tyrosine kinase domain, was expressed. Lane m, 1kb Plus; Lane 1, negative control (no cDNA); Lane 2 and 3, negative control (reactive lymph node cDNA); Lane 4, positive control (normal brain cDNA); Lane 5, cDNA from case 1. (B) Schematic representation of the identified fusion transcripts. The DNA and protein sequence at the fusion border (indicated by the arrowhead) are also shown. TK: tyrosine kinase, NP: nucleoplasmin. (C) Case 1: Nested RT-PCR with forward primers in SEC31A and reverse primers in ALK showed presence of the SEC31A-ALK fusion in case 1, but not in normal liver and reactive lymph node cDNA (upper figure). Lane m, 1kb Plus; Lane 1, negative control (no cDNA); Lane 2, negative control (normal liver cDNA); Lane 3, negative control (reactive lymph node cDNA); Lane 4, cDNA from case 1. An electropherogram with the SEC31A-ALK fusion sequence of the PCR product is also shown (lower figure). (D) Case 2: Nested RT-PCR with forward primers in NPM1 and reverse primers in ALK showed presence of the NPM1-ALK fusion in case 2, but not in reactive lymph node cDNA (upper figure). Lane 1, negative control (no cDNA); Lane 2, negative control (reactive lymph node cDNA); Lane 3, cDNA from case 2. An electropherogram with the NPM1-ALK fusion sequence of the PCR product is also shown (lower figure). (E) IL3 deprivation of Ba/F3 cells transduced with SEC31A-ALK resulted in transformation to growth factor independent growth. When transduced with the empty pMSCVpuro vector, Ba/F3 cells were not able to survive without IL3 and died. The mean growth ± SEM of three separate measurements over five consecutive days is given. (F) SEC31A-ALK and NPM1-ALK expressing Ba/F3 cells were treated with the indicated concentrations of TAE-684 and cell survival was quantified after 48 h. Cell survival in the absence of TAE-684 (0 nM) was set at 100%. Mean ± SEM of four independent measurements is shown. (G) Western blot analysis showing the effect of TAE-684 treatment on Ba/F3 cells transduced with SEC31A-ALK. In addition to ALK phosphorylation, phosphorylation of ERK1/2, AKT, STAT3 and STAT5 decreased with increasing inhibitor concentrations. Expression of total SEC31A-ALK (138 kDa), ERK1/2, AKT, STAT3 and STAT5 remained unaffected.

Article Snippet: Briefly, the SEC31A and ALK fragments were amplified from human tissue cDNA and cloned into the retroviral pMSCVpuro vector (Clontech).

Techniques: Functional Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control, Positive Control, Sequencing, Transduction, Transformation Assay, Plasmid Preparation, Western Blot

Journal: The American journal of pathology

Article Title: BDNF/TrkB signaling augments smooth muscle cell proliferation in pulmonary hypertension.

doi: 10.1016/j.ajpath.2012.08.028

Figure Lengend Snippet: Figure 1. Ntrk2 is up-regulated in mouse lungs after 24 hours of hypoxia exposure (pO2 0.01). Total RNA from mice lung homogenates was isolated (n 18 per group), direct labeled, and hybridized to 44K 60mer oligonucleotide microarrays (MWG). A: Volcano plot: log2-fold regulation compared to probability of regulation. Genes with log odds values 5 were considered to be regulated; black spots, exemplary genes. B: Functional annotation of regulated genes was performed according to Gene Ontology (GO). Ntrk2 (TrkB) belongs to MAPK signaling pathway. C: A time course of TrkB expression was performed using real-time PCR of lung homogenates harvested 1, 7, or 21 days after hypoxia exposure (n 7 to 9, each). D: Immunohistochemical staining of TrkB and its ligand, BDNF in mouse lung sections. The arrow indicates positive staining. E and F: Real-time PCR analysis of TrkB and BDNF expression in (E) isolated major pulmonary arteries (n 6), and (F) laser-microdissected intrapulmonary vessels (40 vessels per animal, n 4), from mice exposed to 21 days of hypoxia or normoxia. G: Expression levels of TrkB and BDNF in mouse PASMC as indicated by real-time PCR; lower CT values represent a more abundant transcript level. H: Immunofluorescence staining of TrkB and BDNF in mouse PASMC. The arrows indicate TrkB localization. Inset, negative control staining. *P 0.05.

Article Snippet: Predesigned, commercially available siRNA against human Egr-1 (siGenomeSMART pool) was purchased from Dharmacon (Chicago, IL), TrkB (siGenomeSMART pool) from Dharmacon and Santa Cruz Biotechnology, siRNA against BDNF (Santa Cruz Biotechnology), control siRNA from Ambion.

Techniques: Isolation, Labeling, Functional Assay, Expressing, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining, Immunofluorescence, Negative Control

Journal: The American journal of pathology

Article Title: BDNF/TrkB signaling augments smooth muscle cell proliferation in pulmonary hypertension.

doi: 10.1016/j.ajpath.2012.08.028

Figure Lengend Snippet: Figure 2. Analysis of TrkB and BDNF expression in rat PAH models. A: Real-time PCR analysis of TrkB and BDNF mRNA expression 14 and 28 days after monocrotaline administration (n 4 per group) in lung homogenates. B: Immunohistochemical staining of TrkB and BDNF in monocrotaline- treated lungs and lungs from control animals. C: Real-time PCR analysis of TrkB and BDNF mRNA expression in lung homogenates in the Su5416 rat model (n 4 controls, n 7 Su5416). n.s., not significant. *P 0.05.

Article Snippet: Predesigned, commercially available siRNA against human Egr-1 (siGenomeSMART pool) was purchased from Dharmacon (Chicago, IL), TrkB (siGenomeSMART pool) from Dharmacon and Santa Cruz Biotechnology, siRNA against BDNF (Santa Cruz Biotechnology), control siRNA from Ambion.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining, Control

Journal: The American journal of pathology

Article Title: BDNF/TrkB signaling augments smooth muscle cell proliferation in pulmonary hypertension.

doi: 10.1016/j.ajpath.2012.08.028

Figure Lengend Snippet: Figure 3. Up-regulation of TrkB and BDNF in human lung tissue from IPAH. A: Colocalization of BDNF and TrkB with SMA as indicated by immunofluorescence staining in donor and IPAH patients. B: Real-time PCR analysis of TrkB and BDNF in laser-microdissected intrapulmonary vessels from donor and IPAH patients. Expression is given in comparison to samples from donor lungs (n 5 per group). C: Real-time PCR analysis of expression levels of TrkB and BDNF in human PASMC; lower CT values represent a more abundant transcript level. D: Immunofluorescence staining of TrkB and BDNF in human PASMC derived from donor and IPAH patients. E: Colocalization of BDNF and TrkB with SMA in PASMC as indicated by immunofluorescence staining, IgG, isotype control staining. *P 0.05.

Article Snippet: Predesigned, commercially available siRNA against human Egr-1 (siGenomeSMART pool) was purchased from Dharmacon (Chicago, IL), TrkB (siGenomeSMART pool) from Dharmacon and Santa Cruz Biotechnology, siRNA against BDNF (Santa Cruz Biotechnology), control siRNA from Ambion.

Techniques: Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Expressing, Comparison, Derivative Assay, Control

Journal: The American journal of pathology

Article Title: BDNF/TrkB signaling augments smooth muscle cell proliferation in pulmonary hypertension.

doi: 10.1016/j.ajpath.2012.08.028

Figure Lengend Snippet: Figure 5. BDNF stimulation leads to MAP kinase activation. A: Time-dependent phosphorylation in response to BDNF of the ERK1/2 kinase with densitometric analysis. B and C: Effects on BDNF signaling by pretreatment with the inhibitors (B) K252a (TrkB) and (C) U0126 (ERK1/2) with densitometric analysis. Equal protein loading was confirmed by immunoblotting against -actin. D: Effects of blocking BDNF antibody on ERK1/2 phosphorylation with densitometric analysis. M(kDa), molecular weight in kilodaltons; n.s., not significant. *P 0.05.

Article Snippet: Predesigned, commercially available siRNA against human Egr-1 (siGenomeSMART pool) was purchased from Dharmacon (Chicago, IL), TrkB (siGenomeSMART pool) from Dharmacon and Santa Cruz Biotechnology, siRNA against BDNF (Santa Cruz Biotechnology), control siRNA from Ambion.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Blocking Assay, Molecular Weight

Journal: The American journal of pathology

Article Title: BDNF/TrkB signaling augments smooth muscle cell proliferation in pulmonary hypertension.

doi: 10.1016/j.ajpath.2012.08.028

Figure Lengend Snippet: Figure 4. Functional role of TrkB and its ligand BDNF in remodeling. Proliferation after BDNF stimulation was assessed by direct [3H] thymidine incorporation (A) (n 6), or immunofluorescence staining of Ki-67 proliferation marker (B). The effect of BDNF on cell apoptosis was measured by annexin V staining (C) and caspase-3 cleavage (D). 5% FCS and staurosporine were used as positive controls for proliferation and apoptosis studies, respectively. *P 0.05.

Article Snippet: Predesigned, commercially available siRNA against human Egr-1 (siGenomeSMART pool) was purchased from Dharmacon (Chicago, IL), TrkB (siGenomeSMART pool) from Dharmacon and Santa Cruz Biotechnology, siRNA against BDNF (Santa Cruz Biotechnology), control siRNA from Ambion.

Techniques: Functional Assay, Immunofluorescence, Staining, Marker

Journal: The American journal of pathology

Article Title: BDNF/TrkB signaling augments smooth muscle cell proliferation in pulmonary hypertension.

doi: 10.1016/j.ajpath.2012.08.028

Figure Lengend Snippet: Figure 6. BDNF regulates and activates Egr-1. A: Induction of early growth-response factor 1 (Egr-1) protein in human PASMC as analyzed by Western blotting. B: BDNF-induced Egr-1 protein localization in PASMC as assessed by immunofluorescence analysis. C: Electrophoretic mobility shift assay for Egr-1 induction and DNA–protein interactions from nuclear extracts prepared from BDNF (10 ng/mL)-treated PASMC, incubated with radiolabeled oligonucleotide containing Egr-1 consensus sequence. Incubation with an excess (10-fold) of an unlabeled probe served as a control for specific binding. PDGF (10 ng/mL) treated cells served as a positive control. D: Egr-1 expression in lung parenchyma of donor and IPAH patients. An Egr-1/ mouse (KO-Egr-1) served as negative control. E: Real-time PCR analysis for Egr-1 expression in IPAH patient and donor laser-microdissected intrapulmonary vessels (n 5). n.s., not significant. *P 0.05.

Article Snippet: Predesigned, commercially available siRNA against human Egr-1 (siGenomeSMART pool) was purchased from Dharmacon (Chicago, IL), TrkB (siGenomeSMART pool) from Dharmacon and Santa Cruz Biotechnology, siRNA against BDNF (Santa Cruz Biotechnology), control siRNA from Ambion.

Techniques: Western Blot, Immunofluorescence, Electrophoretic Mobility Shift Assay, Incubation, Sequencing, Control, Binding Assay, Positive Control, Expressing, Negative Control, Real-time Polymerase Chain Reaction

Journal: The Journal of Neuroscience

Article Title: Brain-Derived Neurotrophic Factor Differentially Regulates Retinal Ganglion Cell Dendritic and Axonal Arborization In Vivo

doi: 10.1523/JNEUROSCI.19-22-09928.1999

Figure Lengend Snippet: Neurotrophins modulate RGC dendritic complexityin vivo. Retinal neurotrophin levels were alteredin vivo by injecting fluorescent microspheres treated with exogenous neurotrophin (BDNF, NGF, NT-3, or NT-4) to increase retinal levels or with function-blocking BDNF antibodies to neutralize endogenous BDNF (anti-BDNF). Control treatments were injections of uncoated, cytochrome c-coated, or nonimmune IgG-coated microspheres. RGCs that developed in BDNF-treated retina, but noncoincident (NC) with the exogenous neurotrophin, were analyzed also. The morphologies of rhodamine–dextran-labeled RGCs were reconstructed from serial optical sections into one plane to quantify morphological parameters that describe dendritic arborization, such as primary dendrite number and branch tip number (see Figs. 3-6). RGCs are depicted in whole mounts viewed from the inner, or vitreal, surface of the retina, and axons are not shown. For control RGCs the averages equaled four to five primary dendrites and 16–20 branch tips, with the highest branch tip order of 4–5 and a 136–175 μm dendrite length. An overall dendritic complexity index (DCI) was calculated by combining several of these morphological measures (see Fig. ​Fig.66 and Materials and Methods for explanation of the calculation). The control RGCs depicted in this figure had DCI values that ranged from 2777 to 2899; NC DCI values ranged from 2417 to 2807; BDNF DCI values ranged between 186 and 276; anti-BDNF DCI values ranged from 5851 to 7429; NGF DCI values ranged from 2705 to 3204; NT-3 DCI values ranged from 2220 to 2588; and NT-4 DCI values ranged from 2213 to 4282. Scale bar, 10 μm.

Article Snippet: Vehicle solution (10% Niu-Twitty), 200 ng/ml BDNF (Amgen), 200 μg/ml of specific function-blocking antibody to BDNF [a gift from J. Carnahan, Amgen; antibody specificity described in Nawa et al. (1995) and Ghosh et al. (1994) ], or 200 μg/ml of nonimmune IgG was pressure-injected into the subpial space overlying the caudal tectum immediately after the first observation.

Techniques: Blocking Assay, Control, Labeling

Journal: The Journal of Neuroscience

Article Title: Brain-Derived Neurotrophic Factor Differentially Regulates Retinal Ganglion Cell Dendritic and Axonal Arborization In Vivo

doi: 10.1523/JNEUROSCI.19-22-09928.1999

Figure Lengend Snippet: Retinal BDNF levels influence RGC dendritic arborization in vivo. Retinal neurotrophin levels were altered in vivo by exposure to exogenous BDNF to increase retinal BDNF or by exposure to function-blocking BDNF antibodies to neutralize endogenous BDNF during RGC dendritic elaboration. RGCs noncoincident (NC) with BDNF-coated microspheres as well as RGCs coincident with control microspheres were analyzed also. Primary dendrite extension was evaluated by determining the number of primary dendrites per RGC (A). Secondary dendritic branching was evaluated by determining the total number of branch tips per RGC (B) as well as the number of branch tips per primary dendrite (C). The extent of dendritic elaboration also was compared by measuring the total dendritic arbor length (D). Primary dendritic number (A), number of dendritic branch tips (B, C), and total arbor length (D) were decreased significantly by exogenous BDNF and increased significantly by neutralizing BDNF. RGCs not coincident (NC) with BDNF-coated microspheres did not differ from controls in any of these morphological parameters. Plotted values represent the averages from five independent experiments; error bars indicate ± SEM. Numbers within thebars indicate the total number (n) of RGCs analyzed for each treatment. ***p < 0.001 as compared with control.

Article Snippet: Vehicle solution (10% Niu-Twitty), 200 ng/ml BDNF (Amgen), 200 μg/ml of specific function-blocking antibody to BDNF [a gift from J. Carnahan, Amgen; antibody specificity described in Nawa et al. (1995) and Ghosh et al. (1994) ], or 200 μg/ml of nonimmune IgG was pressure-injected into the subpial space overlying the caudal tectum immediately after the first observation.

Techniques: In Vivo, Blocking Assay, Control