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Anti-Mouse IgG (whole molecule)-Peroxidase antibody produced in rabbit

RRID:AB_258431

Antibody ID

AB_258431

Target Antigen

Mouse IgG (whole molecule)-Peroxidase antibody produced in rabbit mouse

Proper Citation

(Sigma-Aldrich Cat# A9044, RRID:AB_258431)

Clonality

polyclonal antibody

Comments

Vendor recommendations: IgG immunoblotting (chemiluminescent): 1:80,000-160,000, immunohistochemistry (formalin-fixed, paraffin-embedded sections): 1:200, direct ELISA: 1:40,000; ELISA; Immunohistochemistry; Western Blot

Host Organism

rabbit

Vendor

Sigma-Aldrich

A Genetic Tool to Track Protein Aggregates and Control Prion Inheritance.

  • Newby GA
  • Cell
  • 2017 Nov 2

Literature context:


Abstract:

Protein aggregation is a hallmark of many diseases but also underlies a wide range of positive cellular functions. This phenomenon has been difficult to study because of a lack of quantitative and high-throughput cellular tools. Here, we develop a synthetic genetic tool to sense and control protein aggregation. We apply the technology to yeast prions, developing sensors to track their aggregation states and employing prion fusions to encode synthetic memories in yeast cells. Utilizing high-throughput screens, we identify prion-curing mutants and engineer "anti-prion drives" that reverse the non-Mendelian inheritance pattern of prions and eliminate them from yeast populations. We extend our technology to yeast RNA-binding proteins (RBPs) by tracking their propensity to aggregate, searching for co-occurring aggregates, and uncovering a group of coalescing RBPs through screens enabled by our platform. Our work establishes a quantitative, high-throughput, and generalizable technology to study and control diverse protein aggregation processes in cells.

Funding information:
  • NIAID NIH HHS - DP2 AI131083()
  • NIGMS NIH HHS - R01 GM056350()

The effects of prenatal H1N1 infection at E16 on FMRP, glutamate, GABA, and reelin signaling systems in developing murine cerebellum.

  • Fatemi SH
  • J. Neurosci. Res.
  • 2017 Nov 16

Literature context:


Abstract:

Prenatal viral infection has been identified as a potential risk factor for the development of neurodevelopmental disorders such as schizophrenia and autism. Additionally, dysfunction in gamma-aminobutyric acid, Reelin, and fragile X mental retardation protein (FMRP)-metabotropic glutamate receptor 5 signaling systems has also been demonstrated in these two disorders. In the current report, we have characterized the developmental profiles of selected markers for these systems in cerebella of mice born to pregnant mice infected with human influenza (H1N1) virus on embryonic day 16 or sham-infected controls using SDS-PAGE and Western blotting techniques and evaluated the presence of abnormalities in the above-mentioned markers during brain development. The cerebellum was selected in light of emerging evidence that it plays roles in learning, memory, and emotional processing-all of which are disrupted in autism and schizophrenia. We identified unique patterns of gene and protein expression at birth (postnatal day 0 [P0]), childhood (P14), adolescence (P35), and young adulthood (P56) in both exposed and control mouse progeny. We also identified significant differences in protein expression for FMRP, very-low-density lipoprotein receptor, and glutamic acid decarboxylase 65 and 67 kDa proteins at specific postnatal time points in cerebella of the offspring of exposed mice. Our results provide evidence of disrupted FMRP, glutamatergic, and Reelin signaling in the exposed mouse offspring that explains the multiple brain abnormalities observed in this animal model. © 2016 Wiley Periodicals, Inc.

Epitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication.

  • Courtney DG
  • Cell Host Microbe
  • 2017 Sep 13

Literature context:


Abstract:

Many viral RNAs are modified by methylation of the N6 position of adenosine (m6A). m6A is thought to regulate RNA splicing, stability, translation, and secondary structure. Influenza A virus (IAV) expresses m6A-modified RNAs, but the effects of m6A on this segmented RNA virus remain unclear. We demonstrate that global inhibition of m6A addition inhibits IAV gene expression and replication. In contrast, overexpression of the cellular m6A "reader" protein YTHDF2 increases IAV gene expression and replication. To address whether m6A residues modulate IAV RNA function in cis, we mapped m6A residues on the IAV plus (mRNA) and minus (vRNA) strands and used synonymous mutations to ablate m6A on both strands of the hemagglutinin (HA) segment. These mutations inhibited HA mRNA and protein expression while leaving other IAV mRNAs and proteins unaffected, and they also resulted in reduced IAV pathogenicity in mice. Thus, m6A residues in IAV transcripts enhance viral gene expression.

Funding information:
  • NCI NIH HHS - T32 CA009111()
  • NIAID NIH HHS - R21 AI130574()
  • NIGMS NIH HHS - T32 GM007184()

Synaptic Activity and Muscle Contraction Increases PDK1 and PKCβI Phosphorylation in the Presynaptic Membrane of the Neuromuscular Junction.

  • Hurtado E
  • Front Mol Neurosci
  • 2017 Sep 11

Literature context:


Abstract:

Conventional protein kinase C βI (cPKCβI) is a conventional protein kinase C (PKC) isoform directly involved in the regulation of neurotransmitter release in the neuromuscular junction (NMJ). It is located exclusively at the nerve terminal and both synaptic activity and muscle contraction modulate its protein levels and phosphorylation. cPKCβI molecular maturation includes a series of phosphorylation steps, the first of which is mediated by phosphoinositide-dependent kinase 1 (PDK1). Here, we sought to localize PDK1 in the NMJ and investigate the hypothesis that synaptic activity and muscle contraction regulate in parallel PDK1 and cPKCβI phosphorylation in the membrane fraction. To differentiate the presynaptic and postsynaptic activities, we abolished muscle contraction with μ-conotoxin GIIIB (μ-CgTx-GIIIB) in some experiments before stimulation of the phrenic nerve (1 Hz, 30 min). Then, we analyzed total and membrane/cytosol fractions of skeletal muscle by Western blotting. Results showed that PDK1 is located exclusively in the nerve terminal of the NMJ. After nerve stimulation with and without coincident muscle contraction, total PDK1 and phosphorylated PDK1 (pPDK1) protein levels remained unaltered. However, synaptic activity specifically enhanced phosphorylation of PDK1 in the membrane, an important subcellular location for PDK1 function. This increase in pPDK1 coincides with a significant increase in the phosphorylation of its substrate cPKCβI also in the membrane fraction. Moreover, muscle contraction maintains PDK1 and pPDK1 but increases cPKCβI protein levels and its phosphorylation. Thus, even though PDK1 activity is maintained, pcPKCβI levels increase in concordance with total cPKCβI. Together, these results indicate that neuromuscular activity could induce the membrane targeting of pPDK1 in the nerve terminal of the NMJ to promote the phosphorylation of the cPKCβI, which is involved in ACh release.

Mechanism of Substrate Translocation in an Alternating Access Transporter.

  • Latorraca NR
  • Cell
  • 2017 Mar 23

Literature context:


Abstract:

Transporters shuttle molecules across cell membranes by alternating among distinct conformational states. Fundamental questions remain about how transporters transition between states and how such structural rearrangements regulate substrate translocation. Here, we capture the translocation process by crystallography and unguided molecular dynamics simulations, providing an atomic-level description of alternating access transport. Simulations of a SWEET-family transporter initiated from an outward-open, glucose-bound structure reported here spontaneously adopt occluded and inward-open conformations. Strikingly, these conformations match crystal structures, including our inward-open structure. Mutagenesis experiments further validate simulation predictions. Our results reveal that state transitions are driven by favorable interactions formed upon closure of extracellular and intracellular "gates" and by an unfavorable transmembrane helix configuration when both gates are closed. This mechanism leads to tight allosteric coupling between gates, preventing them from opening simultaneously. Interestingly, the substrate appears to take a "free ride" across the membrane without causing major structural rearrangements in the transporter.

Funding information:
  • NIGMS NIH HHS - R01 GM117108()

Synuclein expression in the lizard Anolis carolinensis.

  • Toni M
  • J. Comp. Physiol. A Neuroethol. Sens. Neural. Behav. Physiol.
  • 2016 Aug 22

Literature context:


Abstract:

The synuclein (syn) family comprises three proteins: α-, β- and γ-syns. In humans, they are involved in neurodegenerative diseases such as Parkinson's disease and in tumors. Members of the syn family were sequenced in representative species of all vertebrates and the comparative analysis of amino acid sequences suggests that syns are evolutionarily conserved, but information about their expression in vertebrate lineages is still scarce and completely lacking in reptiles. In this study, the expression of genes coding for α-, β- and γ-syns was analyzed in the green lizard Anolis carolinensis by semiquantitative RT-PCR and Western blot. Results demonstrate good expression levels of the three syns in the lizard nervous system, similarly to human syns. This, together with the high identity between lizard and human syns, suggests that these proteins fulfill evolutionarily conserved functions. However, differences between lizard and humans in the expression of syn variants (two different variants of γ-syn were detected in A. carolinensis) and differences in some amino acids in key positions for the regulation of protein conformation and affinity for lipid and metal ions also suggest that these proteins may have acquired different functional specializations in the two lineages.

Funding information:
  • NEI NIH HHS - R01 EY025933(United States)
  • NIMH NIH HHS - R21 MH098506(United States)

An optimized protocol for expression and purification of murine perforin in insect cells.

  • Naneh O
  • J. Immunol. Methods
  • 2015 Nov 17

Literature context:


Abstract:

Perforin (PFN) is one of the most important protein effectors of the immune system. It is produced by cytotoxic T lymphocytes and natural killer cells and helps with the clearance of virus-infected and tumor cells. PFN is a pore-forming protein that readily binds to the lipid membranes of target cells, oligomerizes at the cell surface and forms transmembrane pores that allow passage of ions and other larger molecules. Its characterization was hindered in the past by a lack of efficient and reliable expression systems that would result in pure and functional product. In this paper we present optimization of PFN expression in a baculovirus expression system. We optimized several parameters of murine PFN (mPFN) expression and purification and showed that the expressed product is pure and hemolytically active and that it forms pores in the plasma membranes of K562 cells. We could also observe circular pores formed on liposome membranes by cryo-electron microscopy (cryo-EM). Our protocol opens the door for further biochemical and biophysical assessment of PFN properties and interactions with small ligands and lipid membranes.

Localization of α-synuclein in teleost central nervous system: immunohistochemical and Western blot evidence by 3D5 monoclonal antibody in the common carp, Cyprinus carpio.

  • Vaccaro R
  • J. Comp. Neurol.
  • 2015 May 1

Literature context:


Abstract:

Alpha synuclein (α-syn) is a 140 amino acid vertebrate-specific protein, highly expressed in the human nervous system and abnormally accumulated in Parkinson's disease and other neurodegenerative disorders, known as synucleinopathies. The common occurrence of α-syn aggregates suggested a role for α-syn in these disorders, although its biological activity remains poorly understood. Given the high degree of sequence similarity between vertebrate α-syns, we investigated this proteins in the central nervous system (CNS) of the common carp, Cyprinus carpio, with the aim of comparing its anatomical and cellular distribution with that of mammalian α-syn. The distribution of α-syn was analyzed by semiquantitative western blot, immunohistochemistry, and immunofluorescence by a novel monoclonal antibody (3D5) against a fully conserved epitope between carp and human α-syn. The distribution of 3D5 immunoreactivity was also compared with that of choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), and serotonin (5HT) by double immunolabelings. The results showed that a α-syn-like protein of about 17 kDa is expressed to different levels in several brain regions and in the spinal cord. Immunoreactive materials were localized in neuronal perikarya and varicose fibers but not in the nucleus. The present findings indicate that α-syn-like proteins may be expressed in a few subpopulations of catecholaminergic and serotoninergic neurons in the carp brain. However, evidence of cellular colocalization 3D5/TH or 3D5/5HT was rare. Differently, the same proteins appear to be coexpressed with ChAT by cholinergic neurons in several motor and reticular nuclei. These results sustain the functional conservation of the α-syn expression in cholinergic systems and suggest that α-syn modulates similar molecular pathways in phylogenetically distant vertebrates.