mouse anti human cd8β primary antibody  (Bio-Rad)

Journal:

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8??

doi: 10.1128/JVI.79.17.11422-11433.2005

Figure Lengend Snippet: Flow cytometric analysis of Nef-mediated receptor down-regulation and internalization in retrovirally transduced SupT1 cells. SupT1 cells were transduced with an inducible NA-7.ER construct. At time zero, 4-hydroxytamoxifen (1 μM) was added to the culture medium. (A) Percent down-regulation was calculated, as described in the Materials and Methods. CD4-allophycocyanin (solid line) and CD8β-phycoerythrin (dashed line) were measured as a function of time. (B) The figure shows the percentage of CD4, CD8αβ, and CD28 molecules internalized by HIV-1 NA-7.ER, calculated as described in Materials and Methods. In each graph, Nef-positive (EGFP expressing Nef, solid line) and Nef-negative (EGFP not expressing Nef, dashed line) cells are depicted. The EGFP ranges used for calculation are indicated in Fig. ​Fig.1A1A.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8β primary antibody (5F2 [ 15 ], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Transduction, Construct, Expressing

Journal:

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8??

doi: 10.1128/JVI.79.17.11422-11433.2005

Figure Lengend Snippet: Mutations in the CD8 β-chain and their effect on endocytosis and down-regulation. Daudi cells were cotransduced with CD8α, wild-type or mutant CD8β, and control or wild-type Nef. (A and C) Alignment of amino acid sequences of wild-type (210*) and mutant CD8 β-chain cytoplasmic tails. An asterisk indicates a stop codon. (A) The bar chart shows the percent down-regulation of (mutant) CD8αβ by HIV-1 Nef alleles NA-7, LAI, and NL4-3. In both B and D the percentage of (mutant) CD8αβ molecules internalized by wild-type HIV-1 NA-7 is shown, including in both the same data for a Nef-negative construct as a control (wild-type Nef−). (C) The bar chart represents the percentage of (mutant) CD8αβ down-regulation after transduction with either control virus or wild-type Nef NL4-3. Each bar represents a (mutant) CD8 β-chain, as indicated by the changed amino acid sequence compared with CD8αβ. Percent down-regulation and internalization were calculated as described in Materials and Methods, using the ranges indicated in Fig. ​Fig.1A.1A. In A, B, and D, mean values are shown and standard deviations are calculated from the data generated from three independent experiments. In B the results for NL4-3 are representative of the results with HIV-1 alleles NA-7 and LAI.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8β primary antibody (5F2 [ 15 ], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Mutagenesis, Construct, Transduction, Sequencing, Generated

Journal:

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8??

doi: 10.1128/JVI.79.17.11422-11433.2005

Figure Lengend Snippet: Chimeric constructs. Daudi cells were retrovirally transduced with the CD8α(EC-TM)-CD8α(IC) chimera (cyt tail) or a CD8α(EC-TM)-CD8β(IC) chimera (cyt tail), using bicistronic constructs with ΔNGFR as the reporter. Bivariate dot plots are gated on ΔNGFR-positive cells, at day 2 after transduction of these cells with control virus, HIV-1 Nef (NA-7 allele), and SIV Nef (mac239), using bicistronic constructs with EGFP as the reporter. CD8α-phycoerythrin versus EGFP expression is shown.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8β primary antibody (5F2 [ 15 ], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Construct, Transduction, Expressing

Journal:

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8??

doi: 10.1128/JVI.79.17.11422-11433.2005

Figure Lengend Snippet: Confocal images of 293T cells. Nef.EGFP was detected by direct fluorescence (green, left panels) and CD8β.HA or CD8β by monoclonal antibodies as indicated in Materials and Methods (red, middle panels). Nuclei were visualized by DAPI staining (blue). Right panels show the merged images from Nef.EGFP and CD8β. Areas of colocalization of Nef.EGFP/CD8β are shown in yellow. As indicated, the upper panels show cells expressing wild-type LAI, the middle panels show the LLAA mutant, and alower panels show the PPAA mutant. Scale bars represent 5 μm.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8β primary antibody (5F2 [ 15 ], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Fluorescence, Staining, Expressing, Mutagenesis