MHCD0405

antibody from Invitrogen Antibodies

Targeting: CD4

Provider product page for MHCD0405

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [34]
Comments [0]
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

CD4 protein structure - MHCD0405 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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/

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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