Antibody data
- Antibody Data
- Antigen structure
- References [34]
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- Validations
- Flow cytometry [2]
- Other assay [15]
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- Product number
- MHCD0405 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- CD4 Monoclonal Antibody (S3.5), APC
- Antibody type
- Monoclonal
- Antigen
- Other
- Description
- Allophycocyanin (APC) is a stable and highly soluble phycobiliprotein that provides maximal absorbance and fluorescence without susceptibility to internal or external fluorescence quenching, thus providing exceptional quantum yields and molar extinction coefficients.
- Reactivity
- Human
- Host
- Mouse
- Isotype
- IgG
- Antibody clone number
- S3.5
- Vial size
- 500 µL
- Storage
- 4° C, store in dark
Submitted references Broadly active zinc finger protein-guided transcriptional activation of HIV-1.
High-Throughput Generation of Bipod (Fab × scFv) Bispecific Antibodies Exploits Differential Chain Expression and Affinity Capture.
Structural Basis for Tetherin Antagonism as a Barrier to Zoonotic Lentiviral Transmission.
MLLT3 governs human haematopoietic stem-cell self-renewal and engraftment.
Transcriptional Circuit Fragility Influences HIV Proviral Fate.
Preadaptation of Simian Immunodeficiency Virus SIVsmm Facilitated Env-Mediated Counteraction of Human Tetherin by Human Immunodeficiency Virus Type 2.
Human-Specific Adaptations in Vpu Conferring Anti-tetherin Activity Are Critical for Efficient Early HIV-1 Replication In Vivo.
Interleukin-1 receptor-associated kinase 3 downregulation in peripheral blood mononuclear cells attenuates immunosuppression in sepsis.
Species-specific host factors rather than virus-intrinsic virulence determine primate lentiviral pathogenicity.
TIGIT expressing CD4+T cells represent a tumor-supportive T cell subset in chronic lymphocytic leukemia.
Endocytic sorting motif interactions involved in Nef-mediated downmodulation of CD4 and CD3.
Potent antitumour activity of interleukin-2-Fc fusion proteins requires Fc-mediated depletion of regulatory T-cells.
Medial HOXA genes demarcate haematopoietic stem cell fate during human development.
Modeling HIV-1 Latency in Primary T Cells Using a Replication-Competent Virus.
Vpu-Mediated Counteraction of Tetherin Is a Major Determinant of HIV-1 Interferon Resistance.
A Naturally Occurring rev1-vpu Fusion Gene Does Not Confer a Fitness Advantage to HIV-1.
HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells.
PEITC treatment suppresses myeloid derived tumor suppressor cells to inhibit breast tumor growth.
Nef neutralizes the ability of exosomes from CD4+ T cells to act as decoys during HIV-1 infection.
An HIV-encoded antisense long noncoding RNA epigenetically regulates viral transcription.
Subcutaneous versus intravenous administration of rituximab: pharmacokinetics, CD20 target coverage and B-cell depletion in cynomolgus monkeys.
The transmembrane domain of HIV-1 Vpu is sufficient to confer anti-tetherin activity to SIVcpz and SIVgor Vpu proteins: cytoplasmic determinants of Vpu function.
Human neonatal naive CD4+ T cells have enhanced activation-dependent signaling regulated by the microRNA miR-181a.
Characterization of an HIV-targeted transcriptional gene-silencing RNA in primary cells.
Human tetherin exerts strong selection pressure on the HIV-1 group N Vpu protein.
Helicobacter pylori induces in-vivo expansion of human regulatory T cells through stimulating interleukin-1β production by dendritic cells.
Deletion of cIAP1 and cIAP2 in murine B lymphocytes constitutively activates cell survival pathways and inactivates the germinal center response.
A new protocol for multiple inhalation of IFN-gamma successfully treats MDR-TB: a case study.
Phenotypic and molecular characterization of CD103+ CD4+ T cells in bronchoalveolar lavage from patients with interstitial lung diseases.
Graft-versus-leukemia effect after suicide-gene-mediated control of graft-versus-host disease.
Reduced cell surface expression of CCR5 in CCR5Delta 32 heterozygotes is mediated by gene dosage, rather than by receptor sequestration.
A membrane-proximal basic domain and cysteine cluster in the C-terminal tail of CCR5 constitute a bipartite motif critical for cell surface expression.
Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages.
Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages.
Scott TA, O'Meally D, Grepo NA, Soemardy C, Lazar DC, Zheng Y, Weinberg MS, Planelles V, Morris KV
Molecular therapy. Methods & clinical development 2021 Mar 12;20:18-29
Molecular therapy. Methods & clinical development 2021 Mar 12;20:18-29
High-Throughput Generation of Bipod (Fab × scFv) Bispecific Antibodies Exploits Differential Chain Expression and Affinity Capture.
Nesspor TC, Kinealy K, Mazzanti N, Diem MD, Boye K, Hoffman H, Springer C, Sprenkle J, Powers G, Jiang H, La Porte SL, Ganesan R, Singh S, Zwolak A
Scientific reports 2020 May 5;10(1):7557
Scientific reports 2020 May 5;10(1):7557
Structural Basis for Tetherin Antagonism as a Barrier to Zoonotic Lentiviral Transmission.
Buffalo CZ, Stürzel CM, Heusinger E, Kmiec D, Kirchhoff F, Hurley JH, Ren X
Cell host & microbe 2019 Sep 11;26(3):359-368.e8
Cell host & microbe 2019 Sep 11;26(3):359-368.e8
MLLT3 governs human haematopoietic stem-cell self-renewal and engraftment.
Calvanese V, Nguyen AT, Bolan TJ, Vavilina A, Su T, Lee LK, Wang Y, Lay FD, Magnusson M, Crooks GM, Kurdistani SK, Mikkola HKA
Nature 2019 Dec;576(7786):281-286
Nature 2019 Dec;576(7786):281-286
Transcriptional Circuit Fragility Influences HIV Proviral Fate.
Morton EL, Forst CV, Zheng Y, DePaula-Silva AB, Ramirez NP, Planelles V, D'Orso I
Cell reports 2019 Apr 2;27(1):154-171.e9
Cell reports 2019 Apr 2;27(1):154-171.e9
Preadaptation of Simian Immunodeficiency Virus SIVsmm Facilitated Env-Mediated Counteraction of Human Tetherin by Human Immunodeficiency Virus Type 2.
Heusinger E, Deppe K, Sette P, Krapp C, Kmiec D, Kluge SF, Marx PA, Apetrei C, Kirchhoff F, Sauter D
Journal of virology 2018 Sep 15;92(18)
Journal of virology 2018 Sep 15;92(18)
Human-Specific Adaptations in Vpu Conferring Anti-tetherin Activity Are Critical for Efficient Early HIV-1 Replication In Vivo.
Yamada E, Nakaoka S, Klein L, Reith E, Langer S, Hopfensperger K, Iwami S, Schreiber G, Kirchhoff F, Koyanagi Y, Sauter D, Sato K
Cell host & microbe 2018 Jan 10;23(1):110-120.e7
Cell host & microbe 2018 Jan 10;23(1):110-120.e7
Interleukin-1 receptor-associated kinase 3 downregulation in peripheral blood mononuclear cells attenuates immunosuppression in sepsis.
Xia Q, Zhou Y, Wang X, Fu S
Experimental and therapeutic medicine 2018 Feb;15(2):1586-1593
Experimental and therapeutic medicine 2018 Feb;15(2):1586-1593
Species-specific host factors rather than virus-intrinsic virulence determine primate lentiviral pathogenicity.
Joas S, Parrish EH, Gnanadurai CW, Lump E, Stürzel CM, Parrish NF, Learn GH, Sauermann U, Neumann B, Rensing KM, Fuchs D, Billingsley JM, Bosinger SE, Silvestri G, Apetrei C, Huot N, Garcia-Tellez T, Müller-Trutwin M, Hotter D, Sauter D, Stahl-Hennig C, Hahn BH, Kirchhoff F
Nature communications 2018 Apr 10;9(1):1371
Nature communications 2018 Apr 10;9(1):1371
TIGIT expressing CD4+T cells represent a tumor-supportive T cell subset in chronic lymphocytic leukemia.
Catakovic K, Gassner FJ, Ratswohl C, Zaborsky N, Rebhandl S, Schubert M, Steiner M, Gutjahr JC, Pleyer L, Egle A, Hartmann TN, Greil R, Geisberger R
Oncoimmunology 2017;7(1):e1371399
Oncoimmunology 2017;7(1):e1371399
Endocytic sorting motif interactions involved in Nef-mediated downmodulation of CD4 and CD3.
Manrique S, Sauter D, Horenkamp FA, Lülf S, Yu H, Hotter D, Anand K, Kirchhoff F, Geyer M
Nature communications 2017 Sep 5;8(1):442
Nature communications 2017 Sep 5;8(1):442
Potent antitumour activity of interleukin-2-Fc fusion proteins requires Fc-mediated depletion of regulatory T-cells.
Vazquez-Lombardi R, Loetsch C, Zinkl D, Jackson J, Schofield P, Deenick EK, King C, Phan TG, Webster KE, Sprent J, Christ D
Nature communications 2017 May 12;8:15373
Nature communications 2017 May 12;8:15373
Medial HOXA genes demarcate haematopoietic stem cell fate during human development.
Dou DR, Calvanese V, Sierra MI, Nguyen AT, Minasian A, Saarikoski P, Sasidharan R, Ramirez CM, Zack JA, Crooks GM, Galic Z, Mikkola HK
Nature cell biology 2016 Jun;18(6):595-606
Nature cell biology 2016 Jun;18(6):595-606
Modeling HIV-1 Latency in Primary T Cells Using a Replication-Competent Virus.
Martins LJ, Bonczkowski P, Spivak AM, De Spiegelaere W, Novis CL, DePaula-Silva AB, Malatinkova E, Trypsteen W, Bosque A, Vanderkerckhove L, Planelles V
AIDS research and human retroviruses 2016 Feb;32(2):187-93
AIDS research and human retroviruses 2016 Feb;32(2):187-93
Vpu-Mediated Counteraction of Tetherin Is a Major Determinant of HIV-1 Interferon Resistance.
Kmiec D, Iyer SS, Stürzel CM, Sauter D, Hahn BH, Kirchhoff F
mBio 2016 Aug 16;7(4)
mBio 2016 Aug 16;7(4)
A Naturally Occurring rev1-vpu Fusion Gene Does Not Confer a Fitness Advantage to HIV-1.
Langer SM, Hopfensperger K, Iyer SS, Kreider EF, Learn GH, Lee LH, Hahn BH, Sauter D
PloS one 2015;10(11):e0142118
PloS one 2015;10(11):e0142118
HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells.
Reeder JE, Kwak YT, McNamara RP, Forst CV, D'Orso I
eLife 2015 Oct 21;4
eLife 2015 Oct 21;4
PEITC treatment suppresses myeloid derived tumor suppressor cells to inhibit breast tumor growth.
Gupta P, Wright SE, Srivastava SK
Oncoimmunology 2015 Feb;4(2):e981449
Oncoimmunology 2015 Feb;4(2):e981449
Nef neutralizes the ability of exosomes from CD4+ T cells to act as decoys during HIV-1 infection.
de Carvalho JV, de Castro RO, da Silva EZ, Silveira PP, da Silva-Januário ME, Arruda E, Jamur MC, Oliver C, Aguiar RS, daSilva LL
PloS one 2014;9(11):e113691
PloS one 2014;9(11):e113691
An HIV-encoded antisense long noncoding RNA epigenetically regulates viral transcription.
Saayman S, Ackley A, Turner AW, Famiglietti M, Bosque A, Clemson M, Planelles V, Morris KV
Molecular therapy : the journal of the American Society of Gene Therapy 2014 Jun;22(6):1164-1175
Molecular therapy : the journal of the American Society of Gene Therapy 2014 Jun;22(6):1164-1175
Subcutaneous versus intravenous administration of rituximab: pharmacokinetics, CD20 target coverage and B-cell depletion in cynomolgus monkeys.
Mao CP, Brovarney MR, Dabbagh K, Birnböck HF, Richter WF, Del Nagro CJ
PloS one 2013;8(11):e80533
PloS one 2013;8(11):e80533
The transmembrane domain of HIV-1 Vpu is sufficient to confer anti-tetherin activity to SIVcpz and SIVgor Vpu proteins: cytoplasmic determinants of Vpu function.
Kluge SF, Sauter D, Vogl M, Peeters M, Li Y, Bibollet-Ruche F, Hahn BH, Kirchhoff F
Retrovirology 2013 Mar 20;10:32
Retrovirology 2013 Mar 20;10:32
Human neonatal naive CD4+ T cells have enhanced activation-dependent signaling regulated by the microRNA miR-181a.
Palin AC, Ramachandran V, Acharya S, Lewis DB
Journal of immunology (Baltimore, Md. : 1950) 2013 Mar 15;190(6):2682-91
Journal of immunology (Baltimore, Md. : 1950) 2013 Mar 15;190(6):2682-91
Characterization of an HIV-targeted transcriptional gene-silencing RNA in primary cells.
Turner AM, Ackley AM, Matrone MA, Morris KV
Human gene therapy 2012 May;23(5):473-83
Human gene therapy 2012 May;23(5):473-83
Human tetherin exerts strong selection pressure on the HIV-1 group N Vpu protein.
Sauter D, Unterweger D, Vogl M, Usmani SM, Heigele A, Kluge SF, Hermkes E, Moll M, Barker E, Peeters M, Learn GH, Bibollet-Ruche F, Fritz JV, Fackler OT, Hahn BH, Kirchhoff F
PLoS pathogens 2012 Dec;8(12):e1003093
PLoS pathogens 2012 Dec;8(12):e1003093
Helicobacter pylori induces in-vivo expansion of human regulatory T cells through stimulating interleukin-1β production by dendritic cells.
Mitchell PJ, Afzali B, Fazekasova H, Chen D, Ali N, Powell N, Lord GM, Lechler RI, Lombardi G
Clinical and experimental immunology 2012 Dec;170(3):300-9
Clinical and experimental immunology 2012 Dec;170(3):300-9
Deletion of cIAP1 and cIAP2 in murine B lymphocytes constitutively activates cell survival pathways and inactivates the germinal center response.
Gardam S, Turner VM, Anderton H, Limaye S, Basten A, Koentgen F, Vaux DL, Silke J, Brink R
Blood 2011 Apr 14;117(15):4041-51
Blood 2011 Apr 14;117(15):4041-51
A new protocol for multiple inhalation of IFN-gamma successfully treats MDR-TB: a case study.
Grahmann PR, Braun RK
The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease 2008 Jun;12(6):636-44
The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease 2008 Jun;12(6):636-44
Phenotypic and molecular characterization of CD103+ CD4+ T cells in bronchoalveolar lavage from patients with interstitial lung diseases.
Braun RK, Foerster M, Grahmann PR, Haefner D, Workalemahu G, Kroegel C
Cytometry. Part B, Clinical cytometry 2003 Jul;54(1):19-27
Cytometry. Part B, Clinical cytometry 2003 Jul;54(1):19-27
Graft-versus-leukemia effect after suicide-gene-mediated control of graft-versus-host disease.
Litvinova E, Maury S, Boyer O, Bruel S, Benard L, Boisserie G, Klatzmann D, Cohen JL
Blood 2002 Sep 15;100(6):2020-5
Blood 2002 Sep 15;100(6):2020-5
Reduced cell surface expression of CCR5 in CCR5Delta 32 heterozygotes is mediated by gene dosage, rather than by receptor sequestration.
Venkatesan S, Petrovic A, Van Ryk DI, Locati M, Weissman D, Murphy PM
The Journal of biological chemistry 2002 Jan 18;277(3):2287-301
The Journal of biological chemistry 2002 Jan 18;277(3):2287-301
A membrane-proximal basic domain and cysteine cluster in the C-terminal tail of CCR5 constitute a bipartite motif critical for cell surface expression.
Venkatesan S, Petrovic A, Locati M, Kim YO, Weissman D, Murphy PM
The Journal of biological chemistry 2001 Oct 26;276(43):40133-45
The Journal of biological chemistry 2001 Oct 26;276(43):40133-45
Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages.
Lee B, Sharron M, Montaner LJ, Weissman D, Doms RW
Proceedings of the National Academy of Sciences of the United States of America 1999 Apr 27;96(9):5215-20
Proceedings of the National Academy of Sciences of the United States of America 1999 Apr 27;96(9):5215-20
Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages.
Lee B, Sharron M, Montaner LJ, Weissman D, Doms RW
Proceedings of the National Academy of Sciences of the United States of America 1999 Apr 27;96(9):5215-20
Proceedings of the National Academy of Sciences of the United States of America 1999 Apr 27;96(9):5215-20
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- Detection of human IFN-gamma and TNF-a cytokine-producing human CD4 T cells using the Attune® Acoustic Focusing Cytometer. Red blood cell lysed peripheral blood mononuclear cells were left unstimulated (left column) or stimulated (right column) for 5 hours with phorbol myristate acetate (PMA) and ionomycin in the presence of brefeldin A. Cells were then fixed and permeabilized using the FIX & PERM® Cell Permeabilization Kit (Product # GAS-003 or Product # GAS-004) and stained intracellularly with anti-human CD4 APC (Product # MHCD0405) and either anti-human interferon gamma (IFN-gamma) FITC (Product # MHCIFG01) (A) or anti-human tumor necrosis factor alpha (TNF-a) FITC (Product # A18469) (B). Samples were collected using the Attune® Acoustic Focusing Cytometer (blue/red) with 488 nm excitation and 530/30 nm bandpass emission filter to detect FITC. 633 nm excitation and 660/20 nm bandpass emission filter were used to detect APC.
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- Human peripheral blood lymphocytes were stained using APC of anti-human CD4 monoclonal antibody (clone S3.5). The negative control profiles represent unstained cells.
Supportive validation
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- Figure 1 Functional characterization of Vpus from Cameroonian HIV-1 N strains. ( A ) FACS analysis of 293T cells cotransfected with tetherin, CD4, CD1d or NTB-A expression vectors and pCGCG plasmids expressing eGFP alone (lanes 2 and 3) or together with the indicated vpu allele (lanes 4 to 9). eGFP expression levels used to calculate receptor downmodulation and the mean fluorescence intensities (MFIs) are indicated. ( B ) Effect of various Vpus on infectious virus release. 293T cells were cotransfected with HIV-1 DeltaVpu NL4-3 (2 ug), pCGCG vectors coexpressing eGFP and Vpu (500 ng), and a construct expressing HU tetherin (125 ng). Viral supernatants were obtained two days later and used to measure the quantity of infectious HIV-1 in the culture supernatants by infecting TZM-bl indicator cells. Shown is the infectious virion yield relative to that obtained in the absence of tetherin (100%). The results were confirmed in two independent experiments and each symbol indicates infectious virus yield in the presence of one of the 25 vpu alleles analyzed. ( C-F ) Vpu-dependent reduction of ( C ) tetherin, ( D ) CD4, ( E ) CD1d and ( F ) NTB-A surface expression in 293T cells. Shown is the reduction in the levels of receptor cell surface expression relative to those measured in cells transfected with the eGFP only control vector. Each symbol represents n-fold downmodulation of the indicated receptor molecule by one individual vpu allele examined. Shown are average values derived fr
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- Figure 5 Modulation of CD4 by chimeras between HIV-1 and SIVcpz/gor Vpus. (A) FACS analysis of 293T cells co-transfected with a CD4 expression vector and pCGCG plasmids expressing eGFP alone (eGFPonly) or together with the indicated vpu alleles. A construct expressing NL4-3 Vpu was used as a positive control. ( B-D ) Levels of CD4 surface expression in ( B ) 293T, ( C ) HeLa and ( D ) TZM-bl cells in the presence of the indicated vpu alleles are compared to those measured in the absence of Vpu (100%). Values give averages (+-SEM) derived from three to five independent experiments. The same ranges of eGFP expression were used in all calculations. ( E, F ) Correlation between CD4 cell surface levels on 293T and ( E ) HeLa or ( F ) TZM-bl cells. ( G, H ) Correlation between CD4 and tetherin cell surface levels on ( G ) 293T cells and ( H ) TZM-bl and HeLa cells.
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- Fig 3 Functional activity of Rev1-Vpu expressed from pCG expression plasmids. (A) CMV-IE promoter-based pCG expression vectors containing vpu (left panel) or the rev1-vpu fusion gene (right panel). An enhanced version of the green fluorescent protein (eGFP) is co-expressed via an IRES. (B) Expression of Rev1-Vpu and Vpu in HEK293T cells transfected with the indicated pCG vectors. A Vpu-specific antiserum was used for detection. eGFP was detected to check transfection efficiencies. (C-F) FACS analysis of (C) CD4, (D) tetherin, (E) CD1d or (F) NTB-A receptor modulation by ZM247 Vpu and Rev1-Vpu. HEK293T cells were transfected with expression vectors for the respective surface receptor and Vpu or Rev1-Vpu. 40 h post transfection, surface receptor levels were monitored by two-color flow cytometry. Dot plots indicating the gating strategy are shown in the right panels. Bar diagrams summarizing three to five independent experiments +/- SD are shown on the left (***p
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- Fig 4 Vpu function in isogenic viruses differing only in their ability to express Rev1-Vpu. (A, B) FACS analysis of surface expression levels of (A) CD4 and (B) tetherin on HEK293T cells co-transfected with the indicated proviral constructs and expression vectors for the respective surface molecule. Dot plots indicating the gating strategy are shown in the right panels. Bar diagrams summarizing three to six independent experiments +/- SD are shown on the left. (C) p24 release from HEK293T cells co-transfected with the indicated proviral constructs and increasing amounts of a tetherin expression plasmid. 40 h post transfection, the amounts of cell-associated and cell-free p24 were analyzed by ELISA. Relative release was calculated as ratio of p24 in the supernatant to total p24. The means of at least three independent experiments are shown. (D) Activation of NF-kB by viruses harboring the fusion polymorphism or not. HEK293T cells were co-transfected with the indicated proviral clones, and an NF-kB-responsive firefly luciferase reporter construct. 40 h post transfection, firefly luciferase activity was determined and normalized to the activity of a Gaussia luciferase control plasmid. The means of three independent experiments +/- SD are shown (***p
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- Fig 6 Effect of Rev1-Vpu expression on HIV-1 replication in PBMCs and tonsillar explant cultures. (A) PHA-stimulated PBMCs were infected with adjusted amounts of the indicated viruses. Virus replication was monitored by analyzing RT-activity in the supernatant. The means of three independent experiments +/- SEM are shown. (B, C) Surface expression levels of (B) tetherin and (C) CD4 were determined by flow cytometry at day 3 post infection. Infected cells were identified by intracellular p24 staining after surface staining of CD4 or tetherin. Dot plots indicating the gating strategy are shown in the right panels. Bar diagrams summarizing four to five independent experiments +/- SD are shown on the left. (D) Sequence analyses of viral mixtures. Wt and fs mutant virus stocks were normalized for infectivity, mixed at the indicated ratios, and the rev1-vpu region was sequenced after bulk amplification of cDNA. The lower panels show respective standard curves. The peak fluorescence of the T residue at position 1 (ZM246 wt) and the A residue at position 3 (ZM247 wt) is expressed as a fraction of the total fluorescence (relative peak height). (E) Sequence chromatograms of 1:1 input mixtures and viral cultures 10 days post infection (dpi). Percentages of wt and fs sequences displayed in the right panels were calculated from the standard curves shown in (D). (F) Human tonsil explant cultures were infected and analyzed as described in (A). One representative experiment for one of three
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- Fig. 5 Gain-of-function mutations in HIV-1 Nef for CD3 internalization. HEK293T cells were cotransfected with expression vectors for CD3zeta-CD8 or CD4 and constructs coexpressing the indicated nef alleles and eGFP via an IRES and examined by flow cytometric analysis 2 days post transfection. a Examples for primary FACS data with the gating strategy and the mean fluorescence intensity (MFI) of the stained surface receptor and b overview on Nef proteins examined and their CD3 and CD4 downmodulation activities. Bars on the left illustrate the composition of the Nef chimeras A-T. Domains derived from HIV-1 SF2 and SIV mac239 Nef are highlighted in beige and blue , respectively. Numbers on top indicate the respective amino acid positions. Red arrows indicate the Nef proteins that were selected for further analyses in infected primary cells (see Fig. 6 ). Values on the right are normalized to the nef -deficient vector control (100%) and represent means of two to four independent experiments (+-SEM). Numbers in a provide mean fluorescence intensities. See also Supplementary Figs. 5 and 6
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- Fig. 6 Nef-mediated modulation of cell surface receptors in PBMCs. Flow cytometric analysis of a CD3, b CD69, c CD4, d MHCI, e CXCR4, f CD74 and g CD28 surface expression in PBMCs infected with VSV-G pseudotyped HIV-1 M NL4-3 IRES eGFP recombinants expressing the indicated nef alleles. The eGFP expressing (i.e., infected) population was used to calculate receptor downmodulation. Values were normalized to the nef -deficient control (100%). The mean of three independent infections (three donors) +-SEM is shown in the left column . Examples for primary data with the gating strategy and the MFI of the stained surface receptor are shown on the right
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- Figure 5 Functional comparison of a ""bipod"" BsAb produced using either large-scale ( A ) or high-throughput ( B ) methods. ( A ) Graph of the % CARNAVAL cell lysis vs concentration of antibody overlaid with the relative T cell activation, based on CD25 expression. The BsAb displays an EC 50 for CARNAVAL cell killing of 32 pM. ( B ) The same experiment in ( A ) was performed using the BsAb generated in small scale using high-throughput methods, and it shows the antibody has similar activity, displaying an EC 50 for CARNAVAL cell killing of 23 pM.
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- Figure S3 Down-modulation of CD4 in PBMCs infected with HIV-1 IMCs differing in their vpu coding sequences. PHA-activated PBMCs were transduced with the indicated VSVg-pseudotyped HIV-1 IMCs and examined for CD4 surface expression 3 days later. (A) Examples of primary FACS data. Numbers give mean fluorescence intensities (MFI) of CD4 expression in the HIV-1-infected (p24+) cell population. (B) Levels of surface expression in virally infected (p24+) cells relative to uninfected cells (100%). Each symbol provides the result obtained for one individual PBMC donor. Download Figure S3, PDF file, 0.1 MB
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- Figure 3 Determinants of functional differences between HIV-1 N and SIVcpz Vpus. ( A ) Chimeras between the YBF30 (yellow) and EK505 (green) Vpu proteins analyzed. The hydrophobic TMD, the alpha-helical regions and the position of two serine phosphorylation sites are indicated. Dashes indicate gaps introduced to optimize the alignment. The right panel summarizes the functional activity of the respective wild-type or chimeric Vpu proteins. Abbreviations: -, no; (+) low; +, modest and ++ high activity. The asterisks indicate the Vpus shown in panels B to F . ( B ) Effect of selected wild-type and mutant YBF30 and EK505 Vpu proteins and the NL4-3 Vpu on infectious virus release in the presence of tetherin. Curves represent the average infection values (n = 3) relative to those obtained in the absence of tetherin. ( C ) Interaction between tetherin and Vpu detected by BiFC. 293T cells transfected with plasmids expressing tetherin and the indicated Vpu proteins were examined by confocal microscopy. ( D ) Quantitative BiFC assay. Cells were transfected as described in panel A and analyzed by flow cytometry. Shown is the mean fluorescence intensity (MFI) of Kusabira green relative to the MFI of tetherin (100%). The results represent the means from three independent experiments (+-SD). ( E-H ) Efficiencies of down-modulation of ( E ) tetherin, ( F ) CD4, ( G ) CD1d and ( H ) NTB-A cell surface expression in the presence of constructs expressing the indicate Vpu proteins relative to t
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- Figure 1--figure supplement 4. Tat-induced transcriptome changes are also observed at the protein level. Flow cytometry analysis of Jurkat-GFP and -Tat cell lines induced with doxycycline for 24 hr and stained with CD4-APC and CD69-PE antibodies or unstained (negative control) to monitor levels of CD4 and CD69 proteins expressed at the cell surface. Note the increase in the CD69+ population in the presence of Tat. GFP, green fluorescent protein. DOI: http://dx.doi.org/
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- Figure 1. Genomic domains occupied and regulated by Tat in CD4+ T cells. ( A ) Western blot of Jurkat-GFP and -Tat cell lines treated (+) or not (-) with DOX using the indicated antibodies. ( B ) Genome-wide distribution of Tat across the human genome. ( C ) Integration of the FLAG ChIP-seq and RNA-seq datasets defines a set of genes directly regulated by Tat. ( D ) Validation of the RNA-seq dataset using qRT-PCR on the indicated TSG, TDG or non-target genes as negative controls (mean +- SEM; n = 3). ( E ) Individual tracks showing FLAG ChIP-seq and the corresponding RNA-seq dataset in the GFP and Tat cell lines. ( F ) Functional annotation of biological processes enriched at TSG and TDG. This figure is associated with Figure 1--figure supplements 1 - 10 . Direct targets, genes directly bound and regulated by Tat; ChIP-seq, chromatin immunoprecipitation sequencing; DOX, doxycycline; GFP, green fluorescent protein; RNA-seq, RNA sequencing; qRT-PCR, quantitative real time polymerase chain reaction; TDG, Tat downregulated genes; TSG, Tat stimulated genes; TSS, transcription start site. DOI: http://dx.doi.org/ Figure 1--figure supplement 1. Tat protein expression levels in the Jurkat Tat-SF model matches the levels of Tat detected during HIV infection. Western blot (FLAG and Tat) of Jurkat Tat-SF cell line treated with (+) or without (-) DOX, and Jurkat cells latently infected with HIV (clone E4) induced with (+) or without (-) TNF-alpha for 24 hr. A beta-actin western blot is sh