DOCSLIB.ORG

Characterizing the Chlamydia Trachomatis Inclusion Membrane Proteome

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Characterizing the Chlamydia Trachomatis Inclusion Membrane Proteome Characterizing the Chlamydia trachomatis inclusion membrane proteome Mary Dickinson A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2019 Reading committee Kevin Hybiske, Chair Lee Ann Campbell David Sherman Program Authorized to Offer Degree: Pathobiology ©Copyright 2019 Mary Dickinson ii University of Washington Abstract Characterizing the Chlamydia trachomatis inclusion membrane proteome Mary Dickinson Chair of the Supervisory Committee: Kevin Hybiske Pathobiology Program, Department of Medicine Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection, responsible for millions of infections each year. Despite this high prevalence, the elucidation of the molecular mechanisms of Chlamydia pathogenesis has been difficult due to limitations in genetic tools and its intracellular developmental cycle. Within a host epithelial cell, chlamydiae replicate within a vacuole called the inclusion. Many Chlamydia–host interactions are thought to be mediated by the Inc family of type III secreted proteins that are anchored in the inclusion membrane, but their array of host targets are largely unknown. To investigate how the inclusion membrane proteome changes over the course of an infected cell, we have adapted the APEX system of proximity-dependent biotinylation. APEX is capable of specifically labeling proteins within a 20 nm radius in living cells. We transformed C. trachomatis to express the enzyme APEX fused to known inclusion membrane proteins, allowing biotinylation and purification of inclusion-associated proteins. Using quantitative mass spectrometry against APEX labeled samples, we identified over 400 proteins associated with the inclusion membrane at early, middle, and late stages of epithelial cell infection. This system was iii sensitive enough to detect inclusion interacting proteins early in the developmental cycle, at 8 hours post infection, a previously intractable time point. A spreadsheet of the proteins identified by APEX at each time point is included as the supplemental file Table S2-1. Mass spectrometry analysis revealed a novel, early association between C. trachomatis inclusions and endoplasmic reticulum exit sites (ERES), functional regions of the ER where COPII-coated vesicles originate. Pharmacological disruption of ERES function severely restricted early chlamydial growth and the development of infectious progeny. Depletion of certain ERES proteins with RNA interference also reduced formation of infectious progeny. Most ERES proteins tested localized to small punctae that sometimes colocalized with the inclusion membrane, but two ERES proteins that were identified by APEX, PDCD6 and TFG, accumulated on the inclusion membrane in certain conditions. When intracellular calcium was elevated, PDCD6 strongly associated with the inclusion membrane. A truncated form of TFG accumulated on the inclusion membrane. Unlike other ERES proteins tested, depletion of PDCD6 caused enhanced C. trachomatis infectious progeny formation. The work in this dissertation shows that APEX is a powerful in situ approach for identifying critical protein interactions on the membranes of pathogen-containing vacuoles. Furthermore, the data derived from proteomic mapping of Chlamydia inclusions has illuminated an important functional role for ERES in promoting chlamydial developmental growth. iv Table of Contents Table of Contents .................................................................................................... 1 List of Figures ........................................................................................................ 4 List of Tables .......................................................................................................... 6 Acknowledgements ................................................................................................ 7 Chapter 1 – Introduction ........................................................................................ 8 Chlamydia trachomatis disease burden and developmental growth ........... 8 Inclusion development and nutrient acquisition .......................................... 9 Chlamydia genetic manipulation ................................................................ 12 Chlamydia-host interactions ...................................................................... 14 Proteomics of inclusion-interacting proteins .............................................. 15 Dissertation Summary ................................................................................ 17 Chapter 2 – Identifying novel proteins associated with the inclusion membrane using proximity dependent biotinylation ..............................................................18 Introduction ................................................................................................18 Results ....................................................................................................... 20 Development of the APEX in situ proximity labeling system for C. trachomatis ........... 20 Identification of inclusion interacting proteins by quantitative mass spectrometry .....25 Functional validation of targets recruited to early inclusions ......................................... 30 Discussion .................................................................................................. 33 Materials and Methods ............................................................................... 36 Antibodies and reagents....................................................................................................... 36 Cell culture and Chlamydia infections ................................................................................ 36 Plasmid constructs and generation of transformed Chlamydia strains .......................... 37 Biotin-phenol labeling in live cells ...................................................................................... 38 Protein purification for mass spectrometry ....................................................................... 38 Mass spectrometry and bioinformatic analysis of proteomic data ................................. 40 siRNA transfections .............................................................................................................. 42 Quantitative PCR analysis ................................................................................................... 42 Immunofluorescence microscopy ........................................................................................ 43 Western blot analysis ........................................................................................................... 43 Inclusion forming unit analysis .......................................................................................... 44 1 Supplemental Information ......................................................................... 45 Chapter 3 – Endoplasmic reticulum exit sites are required for Chlamydia trachomatis growth ............................................................................................. 48 Introduction ............................................................................................... 48 Results ....................................................................................................... 50 Recruitment of ERES proteins Sec16 and Sec31 to C. trachomatis inclusions ................ 50 Chemical inhibition of ERES cargo loading restricts C. trachomatis developmental growth ..................................................................................................................................... 53 Chemical inhibition of ERES cargo sorting reduces Chlamydia growth ......................... 55 Depletion of ERES regulatory proteins disrupts Chlamydia growth ............................... 61 Discussion .................................................................................................. 63 Materials and Methods ............................................................................... 66 Antibodies and reagents....................................................................................................... 66 Cell culture and Chlamydia infections ................................................................................ 66 Plasmid constructs ................................................................................................................. 67 siRNA and plasmid transfections ......................................................................................... 67 Quantitative PCR analysis ................................................................................................... 68 Immunofluorescence and live cell microscopy .................................................................. 68 Western blot analysis ........................................................................................................... 70 Inclusion forming unit analysis .......................................................................................... 70 Statistical analysis ................................................................................................................. 71 Supplementary Information ...................................................................... 73 Chapter 4 – ER exit site proteins are recruited to the inclusion membrane and have disparate effects on Chlamydia growth ........................................................ 79 Introduction ..............................................................................................
Load more

Recommended publications
  • Farnesol-Induced Apoptosis in Human Lung Carcinoma Cells Is Coupled to the Endoplasmic Reticulum Stress Response
    Research Article Farnesol-Induced Apoptosis in Human Lung Carcinoma Cells Is Coupled to the Endoplasmic Reticulum Stress Response Joung Hyuck Joo,1 Grace Liao,1 Jennifer B. Collins,2 Sherry F. Grissom,2 and Anton M. Jetten1 1Cell Biology Section, LRB, and 2Microarray Group, Division of Intramural Research, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina Abstract range of fruits and vegetables (9, 10). Each isoprenoid has been Farnesol (FOH) and other isoprenoid alcohols induce apopto- shown to inhibit proliferation and induce apoptosis in a number of sis in various carcinoma cells and inhibit tumorigenesis in neoplastic cell lines from different origins (4, 11–14). In addition, in vivo these isoprenoids have been reported to be effective in chemo- several models. However, the mechanisms by which in vivo they mediate their effects are not yet fully understood. In this prevention and chemotherapy in various cancer models study, we show that FOH is an effective inducer of apoptosis in (10, 12, 15, 16). FOH has been reported to exhibit chemopreventive several lung carcinoma cells, including H460. This induction is effects in colon and pancreas carcinogenesis in rats (9, 17) whereas associated with activation of several caspases and cleavage of phase I and II clinical trials have indicated therapeutic potential poly(ADP-ribose) polymerase (PARP). To obtain insight into for POH (16, 18). The mechanisms by which these isoprenoids induce these effects are not yet fully understood. Isoprenoids have the mechanism involved in FOH-induced apoptosis, we compared the gene expression profiles of FOH-treated and been reported to inhibit posttranslational protein prenylation (19) control H460 cells by microarray analysis.
    [Show full text]
  • Multi-Targeted Mechanisms Underlying the Endothelial Protective Effects of the Diabetic-Safe Sweetener Erythritol
    Multi-Targeted Mechanisms Underlying the Endothelial Protective Effects of the Diabetic-Safe Sweetener Erythritol Danie¨lle M. P. H. J. Boesten1*., Alvin Berger2.¤, Peter de Cock3, Hua Dong4, Bruce D. Hammock4, Gertjan J. M. den Hartog1, Aalt Bast1 1 Department of Toxicology, Maastricht University, Maastricht, The Netherlands, 2 Global Food Research, Cargill, Wayzata, Minnesota, United States of America, 3 Cargill RandD Center Europe, Vilvoorde, Belgium, 4 Department of Entomology and UCD Comprehensive Cancer Center, University of California Davis, Davis, California, United States of America Abstract Diabetes is characterized by hyperglycemia and development of vascular pathology. Endothelial cell dysfunction is a starting point for pathogenesis of vascular complications in diabetes. We previously showed the polyol erythritol to be a hydroxyl radical scavenger preventing endothelial cell dysfunction onset in diabetic rats. To unravel mechanisms, other than scavenging of radicals, by which erythritol mediates this protective effect, we evaluated effects of erythritol in endothelial cells exposed to normal (7 mM) and high glucose (30 mM) or diabetic stressors (e.g. SIN-1) using targeted and transcriptomic approaches. This study demonstrates that erythritol (i.e. under non-diabetic conditions) has minimal effects on endothelial cells. However, under hyperglycemic conditions erythritol protected endothelial cells against cell death induced by diabetic stressors (i.e. high glucose and peroxynitrite). Also a number of harmful effects caused by high glucose, e.g. increased nitric oxide release, are reversed. Additionally, total transcriptome analysis indicated that biological processes which are differentially regulated due to high glucose are corrected by erythritol. We conclude that erythritol protects endothelial cells during high glucose conditions via effects on multiple targets.
    [Show full text]
  • 1 Supporting Information for a Microrna Network Regulates
    Supporting Information for A microRNA Network Regulates Expression and Biosynthesis of CFTR and CFTR-ΔF508 Shyam Ramachandrana,b, Philip H. Karpc, Peng Jiangc, Lynda S. Ostedgaardc, Amy E. Walza, John T. Fishere, Shaf Keshavjeeh, Kim A. Lennoxi, Ashley M. Jacobii, Scott D. Rosei, Mark A. Behlkei, Michael J. Welshb,c,d,g, Yi Xingb,c,f, Paul B. McCray Jr.a,b,c Author Affiliations: Department of Pediatricsa, Interdisciplinary Program in Geneticsb, Departments of Internal Medicinec, Molecular Physiology and Biophysicsd, Anatomy and Cell Biologye, Biomedical Engineeringf, Howard Hughes Medical Instituteg, Carver College of Medicine, University of Iowa, Iowa City, IA-52242 Division of Thoracic Surgeryh, Toronto General Hospital, University Health Network, University of Toronto, Toronto, Canada-M5G 2C4 Integrated DNA Technologiesi, Coralville, IA-52241 To whom correspondence should be addressed: Email: [email protected] (M.J.W.); yi- [email protected] (Y.X.); Email: [email protected] (P.B.M.) This PDF file includes: Materials and Methods References Fig. S1. miR-138 regulates SIN3A in a dose-dependent and site-specific manner. Fig. S2. miR-138 regulates endogenous SIN3A protein expression. Fig. S3. miR-138 regulates endogenous CFTR protein expression in Calu-3 cells. Fig. S4. miR-138 regulates endogenous CFTR protein expression in primary human airway epithelia. Fig. S5. miR-138 regulates CFTR expression in HeLa cells. Fig. S6. miR-138 regulates CFTR expression in HEK293T cells. Fig. S7. HeLa cells exhibit CFTR channel activity. Fig. S8. miR-138 improves CFTR processing. Fig. S9. miR-138 improves CFTR-ΔF508 processing. Fig. S10. SIN3A inhibition yields partial rescue of Cl- transport in CF epithelia.
    [Show full text]
  • Noelia Díaz Blanco
    Effects of environmental factors on the gonadal transcriptome of European sea bass (Dicentrarchus labrax), juvenile growth and sex ratios Noelia Díaz Blanco Ph.D. thesis 2014 Submitted in partial fulfillment of the requirements for the Ph.D. degree from the Universitat Pompeu Fabra (UPF). This work has been carried out at the Group of Biology of Reproduction (GBR), at the Department of Renewable Marine Resources of the Institute of Marine Sciences (ICM-CSIC). Thesis supervisor: Dr. Francesc Piferrer Professor d’Investigació Institut de Ciències del Mar (ICM-CSIC) i ii A mis padres A Xavi iii iv Acknowledgements This thesis has been made possible by the support of many people who in one way or another, many times unknowingly, gave me the strength to overcome this "long and winding road". First of all, I would like to thank my supervisor, Dr. Francesc Piferrer, for his patience, guidance and wise advice throughout all this Ph.D. experience. But above all, for the trust he placed on me almost seven years ago when he offered me the opportunity to be part of his team. Thanks also for teaching me how to question always everything, for sharing with me your enthusiasm for science and for giving me the opportunity of learning from you by participating in many projects, collaborations and scientific meetings. I am also thankful to my colleagues (former and present Group of Biology of Reproduction members) for your support and encouragement throughout this journey. To the “exGBRs”, thanks for helping me with my first steps into this world. Working as an undergrad with you Dr.
    [Show full text]
  • Downloaded from URL: Δ Δ Nated ALG-2 GF122) and GST-ALG-2 GF122 Was Cium.Uhnres.Utoronto.Ca/Vgm
    Inuzuka et al. BMC Structural Biology 2010, 10:25 http://www.biomedcentral.com/1472-6807/10/25 RESEARCH ARTICLE Open Access Molecular basis for defect in Alix-binding by alternatively spliced isoform of ALG-2 (ALG-2ΔGF122) and structural roles of F122 in target recognition Tatsutoshi Inuzuka1, Hironori Suzuki1,2, Masato Kawasaki2, Hideki Shibata1, Soichi Wakatsuki2, Masatoshi Maki1* Abstract Background: ALG-2 (a gene product of PDCD6) belongs to the penta-EF-hand (PEF) protein family and Ca2 +-dependently interacts with various intracellular proteins including mammalian Alix, an adaptor protein in the ESCRT system. Our previous X-ray crystal structural analyses revealed that binding of Ca2+ to EF3 enables the side chain of R125 to move enough to make a primary hydrophobic pocket (Pocket 1) accessible to a short fragment of Alix. The side chain of F122, facing a secondary hydrophobic pocket (Pocket 2), interacts with the Alix peptide. An alternatively spliced shorter isoform, designated ALG-2ΔGF122, lacks Gly121Phe122 and does not bind Alix, but the structural basis of the incompetence has remained to be elucidated. Results: We solved the X-ray crystal structure of the PEF domain of ALG-2ΔGF122 in the Ca2+-bound form and compared it with that of ALG-2. Deletion of the two residues shortened a-helix 5 (a5) and changed the configuration of the R125 side chain so that it partially blocked Pocket 1. A wall created by the main chain of 121- GFG-123 and facing the two pockets was destroyed. Surprisingly, however, substitution of F122 with Ala or Gly, but not with Trp, increased the Alix-binding capacity in binding assays.
    [Show full text]
  • PDCD6 Antibody Order 021-34695924 [email protected] Support 400-6123-828 50Ul [email protected] 100 Ul √ √ Web
    TD7978 PDCD6 Antibody Order 021-34695924 [email protected] Support 400-6123-828 50ul [email protected] 100 uL √ √ Web www.ab-mart.com.cn Description: Calcium sensor that plays a key role in processes such as endoplasmic reticulum (ER)- Golgi vesicular transport, endosomal biogenesis or membrane repair. Acts as an adapter that bridges unrelated proteins or stabilizes weak protein-protein complexes in response to calcium: calcium-binding triggers exposure of apolar surface, promoting interaction with different sets of proteins thanks to 3 different hydrophobic pockets, leading to translocation to membranes. Involved in ER-Golgi transport by promoting the association between PDCD6IP and TSG101, thereby bridging together the ESCRT-III and ESCRT-I complexes. Together with PEF1, acts as calcium-dependent adapter for the BCR(KLHL12) complex, a complex involved in ER-Golgi transport by regulating the size of COPII coats. In response to cytosolic calcium increase, the heterodimer formed with PEF1 interacts with, and bridges together the BCR(KLHL12) complex and SEC31 (SEC31A or SEC31B), promoting monoubiquitination of SEC31 and subsequent collagen export, which is required for neural crest specification. Involved in the regulation of the distribution and function of MCOLN1 in the endosomal pathway. Promotes localization and polymerization of TFG at endoplasmic reticulum exit site. Required for T-cell receptor-, Fas-, and glucocorticoid- induced apoptosis (By similarity). May mediate Ca(2+)-regulated signals along the death pathway: interaction with DAPK1 can accelerate apoptotic cell death by increasing caspase-3 activity. Its role in apoptosis may however be indirect, as suggested by knockout experiments (By similarity). May inhibit KDR/VEGFR2-dependent angiogenesis; the function involves inhibition of VEGF-induced phosphorylation of the Akt signaling pathway.
    [Show full text]
  • Identification of Promoter Regions in the Human Genome by Using a Retroviral Plasmid Library-Based Functional Reporter Gene Assa
    Downloaded from genome.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Methods Identification of Promoter Regions in the Human Genome by Using a Retroviral Plasmid Library-Based Functional Reporter Gene Assay Shirin Khambata-Ford,1,5 Yueyi Liu,2 Christopher Gleason,1 Mark Dickson,3 Russ B. Altman,2 Serafim Batzoglou,4 and Richard M. Myers1,3,6 1Department of Genetics, 2Stanford Medical Informatics, 3Stanford Human Genome Center, Stanford University School of Medicine, Stanford, California 94305, USA; 4Department of Computer Science, Stanford University, Stanford, California 94305, USA Attempts to identify regulatory sequences in the human genome have involved experimental and computational methods such as cross-species sequence comparisons and the detection of transcription factor binding-site motifs in coexpressed genes. Although these strategies provide information on which genomic regions are likely to be involved in gene regulation, they do not give information on their functions. We have developed a functional selection for promoter regions in the human genome that uses a retroviral plasmid library-based system. This approach enriches for and detects promoter function of isolated DNA fragments in an in vitro cell culture assay. By using this method, we have discovered likely promoters of known and predicted genes, as well as many other putative promoter regions based on the presence of features such as CpG islands. Comparison of sequences of 858 plasmid clones selected by this assay with the human genome draft sequence indicates that a significantly higher percentage of sequences align to the 500-bp segment upstream of the transcription start sites of known genes than would be expected from random genomic sequences.
    [Show full text]
  • Proquest Dissertations
    RICE UNIVERSITY Molecular Basis of Gene Dosage Sensitivity by Jianping Chen A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE Doctor of Philosophy APPROVED, THESIS COMMITTEE: Ariel Fernandez, Chair Karl F. Hasselmann Professor Department of Bioengineering Rice University rn'idtfP h.tQsJUW*- Michael W. Deem, John W. Cox Professor Department of Bioengineering Department of Physics and Astronomy Rice Universit ^engineering at Rice University Jiochemistry at Baylor College of Medicine Axxttig- «J^gb/uv Laura Segaton, T.N. Law Assistant Professor Chemical and Biomolecular Engineering Rice University HOUSTON, TEXAS JANUARY 2009 UMI Number: 3362141 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. UMI® UMI Microform 3362141 Copyright 2009 by ProQuest LLC All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT Molecular Basis of Gene Dosage Sensitivity by JianpingChen Deviation of gene expression from normal levels has been associated with diseases. Both under- and overexpression of genes could lead to deleterious biological consequences. Dosage balance has been proposed to be a key issue of determining gene expression pheno- type.
    [Show full text]
  • Identification of Biomarkers of Metastatic Disease in Uveal
    Identification of biomarkers of metastatic disease in uveal melanoma using proteomic analyses Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy Martina Angi June 2015 To Mario, the wind beneath my wings 2 Acknowledgments First and foremost, I would like to acknowledge my primary supervisor, Prof. Sarah Coupland, for encouraging me to undergo a PhD and for supporting me in this long journey. I am truly grateful to Dr Helen Kalirai for being the person I could always turn to, for a word of advice on cell culture as much as on parenting skills. I would also like to acknowledge Prof. Bertil Damato for being an inspiration and a mentor; and Dr Sarah Lake and Dr Joseph Slupsky for their precious advice. I would like to thank Dawn, Haleh, Fidan and Fatima for becoming my family away from home, and the other members of the LOORG for the fruitful discussions and lovely cakes. I would like to acknowledge Prof. Heinrich Heimann and the clinical team at LOOC, especially Sisters Hebbar, Johnston, Hachuela and Kaye, for their admirable dedication to UM patients and for their invaluable support to clinical research. I would also like to thank the members of staff in St Paul’s theatre and Simon Biddolph and Anna Ikin in Pathology for their precious help in sample collection. I am grateful to Dr Rosalind Jenkins who guided my first steps in the mysterious word of proteomics, and to Dr Deb Simpsons and Prof. Rob Beynon for showing me its beauty.
    [Show full text]
  • PDCD6IP (Human) Recombinant Protein (P01)
    PDCD6IP (Human) Recombinant Preparation Method: in vitro wheat germ expression Protein (P01) system Purification: Glutathione Sepharose 4 Fast Flow Catalog Number: H00010015-P01 Storage Buffer: 50 mM Tris-HCI, 10 mM reduced Regulation Status: For research use only (RUO) Glutathione, pH=8.0 in the elution buffer. Product Description: Human PDCD6IP full-length ORF Storage Instruction: Store at -80°C. Aliquot to avoid ( AAH20066, 1 a.a. - 868 a.a.) recombinant protein with repeated freezing and thawing. GST-tag at N-terminal. Entrez GeneID: 10015 Sequence: MATFISVQLKKTSEVDLAKPLVKFIQQTYPSGGEEQAQ Gene Symbol: PDCD6IP YCRAAEELSKLRRAAVGRPLDKHEGALETLLRYYDQIC SIEPKFPFSENQICLTFTWKDAFDKGSLFGGSVKLALA Gene Alias: AIP1, Alix, DRIP4, HP95, MGC17003 SLGYEKSCVLFNCAALASQIAAEQNLDNDEGLKIAAKH YQFASGAFLHIKETVLSALSREPTVDISPDTVGTLSLIM Gene Summary: This gene encodes a protein thought LAQAQEVFFLKATRDKMKDAIIAKLANQAADYFGDAFK to participate in programmed cell death. Studies using QCQYKDTLPKEVFPVLAAKHCIMQANAEYHQSILAKQ mouse cells have shown that overexpression of this QKKFGEEIARLQHAAELIKTVASRYDEYVNVKDFSDKI protein can block apoptosis. In addition, the product of NRALAAAKKDNDFIYHDRVPDLKDLDPIGKATLVKSTP this gene binds to the product of the PDCD6 gene, a VNVPISQKFTDLFEKMVPVSVQQSLAAYNQRKADLVN protein required for apoptosis, in a calcium-dependent RSIAQMREATTLANGVLASLNLPAAIEDVSGDTVPQSIL manner. This gene product also binds to endophilins, TKSRSVIEQGGIQTVDQLIKELPELLQRNREILDESLRLL proteins that regulate membrane shape during DEEEATDNDLRAKFKERWQRTPSNELYKPLRAEGTNF endocytosis.
    [Show full text]
  • Anti-PEF1 / Peflin Antibody (ARG42993)
    Product datasheet [email protected] ARG42993 Package: 100 μl anti-PEF1 / Peflin antibody Store at: -20°C Summary Product Description Rabbit Polyclonal antibody recognizes PEF1 / Peflin Tested Reactivity Hu, Ms, Rat Tested Application FACS, WB Host Rabbit Clonality Polyclonal Isotype IgG Target Name PEF1 / Peflin Antigen Species Human Immunogen Synthetic peptide of Human PEF1 / Peflin. Conjugation Un-conjugated Alternate Names ABP32; PEF1A; Peflin; PEF protein with a long N-terminal hydrophobic domain; Penta-EF hand domain- containing protein 1 Application Instructions Application table Application Dilution FACS 1:20 WB 1:1000 - 1:5000 Application Note * The dilutions indicate recommended starting dilutions and the optimal dilutions or concentrations should be determined by the scientist. Positive Control K562 Calculated Mw 30 kDa Observed Size ~ 31 kDa Properties Form Liquid Purification Affinity purified. Buffer 50 nM Tris-Glycine (pH 7.4), 0.15M NaCl, 0.01% Sodium azide, 40% Glycerol and 0.05% BSA. Preservative 0.01% Sodium azide Stabilizer 40% Glycerol and 0.05% BSA Storage instruction For continuous use, store undiluted antibody at 2-8°C for up to a week. For long-term storage, aliquot and store at -20°C. Storage in frost free freezers is not recommended. Avoid repeated freeze/thaw cycles. Suggest spin the vial prior to opening. The antibody solution should be gently mixed before use. www.arigobio.com 1/2 Note For laboratory research only, not for drug, diagnostic or other use. Bioinformation Gene Symbol PEF1 Gene Full Name penta-EF-hand domain containing 1 Background This gene encodes a calcium-binding protein belonging to the penta-EF-hand protein family.
    [Show full text]
  • Functional Dependency Analysis Identifies Potential Druggable
    cancers Article Functional Dependency Analysis Identifies Potential Druggable Targets in Acute Myeloid Leukemia 1, 1, 2 3 Yujia Zhou y , Gregory P. Takacs y , Jatinder K. Lamba , Christopher Vulpe and Christopher R. Cogle 1,* 1 Division of Hematology and Oncology, Department of Medicine, College of Medicine, University of Florida, Gainesville, FL 32610-0278, USA; yzhou1996@ufl.edu (Y.Z.); gtakacs@ufl.edu (G.P.T.) 2 Department of Pharmacotherapy and Translational Research, College of Pharmacy, University of Florida, Gainesville, FL 32610-0278, USA; [email protected]fl.edu 3 Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610-0278, USA; cvulpe@ufl.edu * Correspondence: [email protected]fl.edu; Tel.: +1-(352)-273-7493; Fax: +1-(352)-273-5006 Authors contributed equally. y Received: 3 November 2020; Accepted: 7 December 2020; Published: 10 December 2020 Simple Summary: New drugs are needed for treating acute myeloid leukemia (AML). We analyzed data from genome-edited leukemia cells to identify druggable targets. These targets were necessary for AML cell survival and had favorable binding sites for drug development. Two lists of genes are provided for target validation, drug discovery, and drug development. The deKO list contains gene-targets with existing compounds in development. The disKO list contains gene-targets without existing compounds yet and represent novel targets for drug discovery. Abstract: Refractory disease is a major challenge in treating patients with acute myeloid leukemia (AML). Whereas the armamentarium has expanded in the past few years for treating AML, long-term survival outcomes have yet to be proven. To further expand the arsenal for treating AML, we searched for druggable gene targets in AML by analyzing screening data from a lentiviral-based genome-wide pooled CRISPR-Cas9 library and gene knockout (KO) dependency scores in 15 AML cell lines (HEL, MV411, OCIAML2, THP1, NOMO1, EOL1, KASUMI1, NB4, OCIAML3, MOLM13, TF1, U937, F36P, AML193, P31FUJ).
    [Show full text]