anti-mcd8α  (Bio X Cell)

Journal: eLife

Article Title: Drosophila mushroom bodies integrate hunger and satiety signals to control innate food-seeking behavior

doi: 10.7554/eLife.35264

Figure Lengend Snippet:

Article Snippet: Antibody , Rat anti-mCD8α , Thermo Fisher Scientific, MA, USA , RRID: AB_10392843 , 1:100.

Techniques: Cider Assay, Paraffin Wax, Isolation, Software

Journal: Cell reports

Article Title: Regulatory T cell differentiation is controlled by αKG-induced alterations in mitochondrial metabolism and lipid homeostasis

doi: 10.1016/j.celrep.2021.109911

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mCD8α- APC-eF780 (clone 53-6.7) , eBioscience/ThermoFisher , Cat# 47-0081-82 RRID: AB_1272185.

Techniques: Flow Cytometry, Recombinant, Saline, XF Assay, Cell Isolation, Reverse Transcription, SYBR Green Assay, RNA HS Assay, Picogreen Assay, Ligation, Sequencing, Staining, Software, ATP Assay, FCAP Assay

Journal: eLife

Article Title: Cytotoxic T-cells mediate exercise-induced reductions in tumor growth

doi: 10.7554/eLife.59996

Figure Lengend Snippet: ( A ) FVB mice were allowed to exercise voluntarily (in running wheels, runners) or left non-exercised (locked running wheels, non-runners) before and after being inoculated subcutaneously with 5 × 10 5 tumor cells of the breast cancer cell line I3TC. ( B ) Mean tumor volume and SEM over time (left) and survival (right). **p<0.01, *p<0.05, Log-rank (Mantel-Cox) survival test. ( C–E ) Flow cytometry determined frequency of lymphocytic populations within I3TC tumor, spleen and lymph nodes (LN) at day 55 after inoculation. *p<0.05 column factor combining all organs in a two-way ANOVA. ns = not significant. ( F–G ) Flow cytometry determined frequency of lymphocytic populations within spleen and I3TC tumors after CD8+ depletion and isotype control. ( H–I ) Same experimental setting as in ( A ) with (αCD8) or without (isotype ctrl) weekly antibody-mediated depletion of CD8+ T cells (arrows). Graphs show mean tumor volume and SEM over time ( F ) and survival ( G ). *p<0.05, Log-rank (Mantel-Cox) survival test.

Article Snippet: For CD8 depletion, 200 μg of antibodies (anti-mCD8α clone 53–6.72, BioXCell or Rat IgG2a Isotype control, clone 2A3, BioXCell) was injected i.p at day 4 post-tumor cell injections and repeated weekly throughout the experiment.

Techniques: Flow Cytometry, Control

Journal: eLife

Article Title: Cytotoxic T-cells mediate exercise-induced reductions in tumor growth

doi: 10.7554/eLife.59996

Figure Lengend Snippet: ( A ) Reference values of central carbon metabolite availability in resting, human adult individuals. ( B ) Proliferation of activated murine CD8 + T cells was assessed with a resazurin assay after 3 days of cell culture. The response is expressed as fold change over the control (0 mM concentration). Error bars represents the standard error of the mean (SEM). CD8 + T cells purified and activated from pooled spleens of multiple mice in four separate experiments ( n = 4). ( C ) Proliferation of activated murine CD8 + T cells in response to increasing concentrations of central carbon metabolites and sodium chloride control. The proliferation of activated murine CD8 + T cells was assessed by using CountBright counting beads on flow cytometry at day 3 of culture. CD8 + T cells purified and activated from four individual mice ( n = 4). ( D ) Flow-cytometry-based CD62L and CD44 expression of live CD8 + T cells expressed as frequency of cells at day 3 of culture with increasing concentrations of central carbon metabolites. CD8 + T cells purified and activated from four individual mice ( n = 4). ( E ) Granzyme B median fluorescence intensity (MFI) at day 3 of culture with increasing concentrations of central carbon metabolites. CD8 + T cells purified and activated from four individual mice ( n = 4). Two-way ANOVA comparing sodium L-lactate and sodium chloride. *p<0.05, **p<0.01. ( F ) ICOS median fluorescence intensity (MFI) at day 3 of culture with increasing concentrations of central carbon metabolites. CD8 + T cells purified and activated from four individual mice ( n = 4). ( G ) CTLA4 median fluorescence intensity (MFI) at day 3 of culture with increasing concentrations of central carbon metabolites. CD8 + T cells purified and activated from four individual mice ( n = 4).

Article Snippet: For CD8 depletion, 200 μg of antibodies (anti-mCD8α clone 53–6.72, BioXCell or Rat IgG2a Isotype control, clone 2A3, BioXCell) was injected i.p at day 4 post-tumor cell injections and repeated weekly throughout the experiment.

Techniques: Resazurin Assay, Cell Culture, Control, Concentration Assay, Purification, Flow Cytometry, Expressing, Fluorescence

Journal: eLife

Article Title: Cytotoxic T-cells mediate exercise-induced reductions in tumor growth

doi: 10.7554/eLife.59996

Figure Lengend Snippet: ( A ) Proliferation of activated murine CD8 + T cells in response to increasing concentrations of central carbon metabolites. The proliferation of activated murine CD8 + T cells was assessed by using CountBright counting beads on flow cytometry at day 3 of culture. ( B ) Flow-cytometry-based CD62L and CD44 expression of live CD8 + T cells expressed as frequency of cells at day 3 of culture with increasing concentrations of central carbon metabolites. Shaded areas represent mean and 95% confidence intervals at 0 mM. ( C ) Granzyme B median fluorescence intensity (MFI) at day 3 of culture with increasing concentrations of central carbon metabolites. Data from one representative experiment on CD8 + T cells purified and activated from pooled spleens of multiple mice. ( D ) ICOS median fluorescence intensity (MFI) at day 3 of culture with increasing concentrations of central carbon metabolites. Data from one representative experiment on CD8 + T cells purified and activated from pooled spleens of multiple mice. ( E ) Cytotoxicity against EL4-OVA tumor cells by OVA-specific OT-I CD8+ T cells activated for 3 days in the presence of 40 mM NaCl or NaLac. Graph represents specific cytotoxicity of n = 3 independent mouse donors at varying effector-to-target ratios. *p<0.01 repeated-measures two-way ANOVA with Sidak’s multiple comparison test.

Article Snippet: For CD8 depletion, 200 μg of antibodies (anti-mCD8α clone 53–6.72, BioXCell or Rat IgG2a Isotype control, clone 2A3, BioXCell) was injected i.p at day 4 post-tumor cell injections and repeated weekly throughout the experiment.

Techniques: Flow Cytometry, Expressing, Fluorescence, Purification, Comparison

Journal: eLife

Article Title: Cytotoxic T-cells mediate exercise-induced reductions in tumor growth

doi: 10.7554/eLife.59996

Figure Lengend Snippet: ( A ) Recipient mice received OT-I CD8+ T-cells followed by vaccination. On day 2 and 3 after vaccination, 10 mg of [U- 13 C 6 ]glucose was introduced to the mice prior to a treadmill exercise. CD8+ T-cells were harvested from the spleen and incorporation of labeled carbons in cellular metabolites was assessed by GC-MS. Data is provided as . ( B ) Fraction of labeled metabolites in CD8+ T-cells 48 hr after vaccination. ( C ) Fraction of labeled metabolites in CD8+ T-cells 72 hr after vaccination. **p<0.01 and ***p<0.001 using a two-way ANOVA with Sidak’s multiple comparison test. Figure 4—source data 1. Glucose derived carbon distribution in CD8+ T cells source data file.

Article Snippet: For CD8 depletion, 200 μg of antibodies (anti-mCD8α clone 53–6.72, BioXCell or Rat IgG2a Isotype control, clone 2A3, BioXCell) was injected i.p at day 4 post-tumor cell injections and repeated weekly throughout the experiment.

Techniques: Labeling, Gas Chromatography-Mass Spectrometry, Comparison, Derivative Assay

Journal: eLife

Article Title: Cytotoxic T-cells mediate exercise-induced reductions in tumor growth

doi: 10.7554/eLife.59996

Figure Lengend Snippet: ( A ) OT-I mice carrying the congenic marker CD45.1 were given access to a locked or moving running wheel for 6 weeks. In parallel, C57Bl/6 (CD45.2) animals were inoculated with 5 × 10 5 ovalbumin (OVA)-expressing B16-F10 (B16-F10-OVA) melanoma and conditioned with 300 mg/kg cyclophosphamide (CPA). 4 × 10 5 OVA-reactive OT-I CD8+ T cells were isolated from running or non-running mice and adoptively transferred into tumor-bearing animals. Peripheral blood was sampled 10 days after adoptive transfer and tumor volume monitored. ( B ) Flow cytometry analysis of OT-I T cell expansion in peripheral blood 10 days after adoptive transfer. Adoptive cells were distinguished from endogenous immune cells by expression of the CD45.1 congenic marker. Histogram shows ICOS surface expression on adoptive and endogenous CD8+ T cells. ( C ) Frequency of adoptive OT-I T cells in peripheral blood. *p<0.05, two-tailed t-test. ns = not significant. ( D ) Median fluorescence intensity (MFI) of ICOS (bottom) of adoptive OT-I T cells in peripheral blood. *p<0.05, two-tailed t-test. ns = not significant. ( E–G ) Mean tumor volume and SEM ( E ), median IQR ( F ) and survival ( G ) over time. ↓ depicts time of OT-I T cell injection *p<0.05, Log-rank (Mantel-Cox) survival test.

Article Snippet: For CD8 depletion, 200 μg of antibodies (anti-mCD8α clone 53–6.72, BioXCell or Rat IgG2a Isotype control, clone 2A3, BioXCell) was injected i.p at day 4 post-tumor cell injections and repeated weekly throughout the experiment.

Techniques: Marker, Expressing, Isolation, Adoptive Transfer Assay, Flow Cytometry, Two Tailed Test, Fluorescence, Injection

Journal: eLife

Article Title: Cytotoxic T-cells mediate exercise-induced reductions in tumor growth

doi: 10.7554/eLife.59996

Figure Lengend Snippet: ( A ) Peripheral blood lactate concentration (as measured with Accutrend Plus) following a single dose of 2 g/kg Sodium L-lactate (NaLac) administered i.p in FVB or C57BL/6J mice. ( B ) Daily doses of PBS or 3 g/kg NaLac were administered i.p to FVB mice for 12 days before subcutaneous inoculation with the breast cancer cell line I3TC. Injections were continued throughout the experiment. Graphs show tumor volume (mean and SEM) over time. *p<0.05, Log-rank (Mantel-Cox) survival test. ( C ) Flow-cytometrybased assessment of tumor infiltrating CD8+ T-cells (CD8+ cells as % of CD45+ cells) in I3TC tumors after daily lactate administration. *p<0.05, two-tailed t-test. ( D ) Flow-cytometry-based assessment of tumor infiltrating GzmB+CD8+ T-cells (% of CD45+ cells) in I3TC tumors after daily lactate administration. Ns = not significant, two-tailed t-test. ( E ) Flow-cytometry-based assessment of GzmB (left panel), PD1 (middle panel), and CTLA4 (right panel) MFI of CD8+ T-cells in I3TC tumors after daily lactate administration. Ns = not significant, two-tailed t-test.

Article Snippet: For CD8 depletion, 200 μg of antibodies (anti-mCD8α clone 53–6.72, BioXCell or Rat IgG2a Isotype control, clone 2A3, BioXCell) was injected i.p at day 4 post-tumor cell injections and repeated weekly throughout the experiment.

Techniques: Concentration Assay, Two Tailed Test, Flow Cytometry

Journal: eLife

Article Title: Cytotoxic T-cells mediate exercise-induced reductions in tumor growth

doi: 10.7554/eLife.59996

Figure Lengend Snippet: ( A ) Daily doses of PBS or 0.5, 1, or 2 g/kg Sodium L-lactate (NaLac) were administered i.p to FVB mice for 12 days before subcutaneous inoculation with 5 × 10 5 cells of the MMTV-PyMT-derived breast cancer cell line I3TC. Daily sodium L-lactate injections were continued throughout the experiment. Graphs show tumor volume (mean and SEM) over time and survival. *p<0.05, Log-rank (Mantel-Cox) survival test. ( B ) Flow cytometric characterization of I3TC tumor infiltrating immune cell populations. **p<0.01, *p<0.05, two-tailed t test. ( C ) Daily doses of PBS or 2 g/kg NaLac was administered i.p to C57BL/6J mice for 12 days before subcutaneous inoculation with 5 × 10 5 cells of the colon cancer cell line MC38. Injections were continued throughout the experiment. Graphs show tumor volume (mean and SEM) over time and survival. **p<0.01, Log-rank (Mantel-Cox) survival test. ( D ) Same experimental setting as in ( A ) with or without weekly antibody-mediated depletion of CD8+ T cells (arrows). Graphs show tumor volume (mean and SEM) over time and survival. ns = not significant, Log-rank (Mantel-Cox) survival test.

Article Snippet: For CD8 depletion, 200 μg of antibodies (anti-mCD8α clone 53–6.72, BioXCell or Rat IgG2a Isotype control, clone 2A3, BioXCell) was injected i.p at day 4 post-tumor cell injections and repeated weekly throughout the experiment.

Techniques: Derivative Assay, Two Tailed Test

Journal: eLife

Article Title: Cytotoxic T-cells mediate exercise-induced reductions in tumor growth

doi: 10.7554/eLife.59996

Figure Lengend Snippet: ( A ) Recipient mice received OT-I CD8+ T-cells followed by vaccination. On day 2 and 3 after vaccination, 10 mg of [U- 13 C 6 ]glucose was introduced to the mice prior to a treadmill exercise. CD8+ T-cells were harvested from the spleen and incorporation of labeled carbons in cellular metabolites was assessed by GC-MS (n = 4). Metabolic diagram showing total glucose fraction in in exercising (EXERCISE) and non-exercising (CTRL) animals at 48 and 72 hr after vaccination. Two-tailed t-test *p<0.05, **p<0.01. ( B ) Metabolic diagram showing labeled glucose fraction in in exercising (EXERCISE) and non-exercising (CTRL) animals at 48 and 72 hr after vaccination. Two-tailed t-test *p<0.05, **p<0.01,.

Article Snippet: For CD8 depletion, 200 μg of antibodies (anti-mCD8α clone 53–6.72, BioXCell or Rat IgG2a Isotype control, clone 2A3, BioXCell) was injected i.p at day 4 post-tumor cell injections and repeated weekly throughout the experiment.

Techniques: Labeling, Gas Chromatography-Mass Spectrometry, Two Tailed Test

Journal: eLife

Article Title: Cytotoxic T-cells mediate exercise-induced reductions in tumor growth

doi: 10.7554/eLife.59996

Figure Lengend Snippet:

Article Snippet: For CD8 depletion, 200 μg of antibodies (anti-mCD8α clone 53–6.72, BioXCell or Rat IgG2a Isotype control, clone 2A3, BioXCell) was injected i.p at day 4 post-tumor cell injections and repeated weekly throughout the experiment.

Techniques: Transgenic Assay, Marker, Derivative Assay, Staining, Software, Plasmid Preparation, Avidin-Biotin Assay

Journal: Nature immunology

Article Title: Negative regulation of CD8 expression via CD8 enhancer-mediated recruitment of the zinc finger protein MAZR

doi: 10.1038/ni1311

Figure Lengend Snippet: Epigenetic changes at the Cd8a and Cd8b1 gene complex in E8I,E8II doubly deficient (Δ1Δ2/Δ1Δ2) thymocytes. (a) Flow cytometric analysis of CD4 and CD8 expression on thymocytes isolated from either +/Δ1Δ2 × Tcra−/− or Δ1Δ2/Δ1Δ2 × Tcra−/− mice as reported previously14. The numbers in the quadrants indicate the percentage of the corresponding thymocyte subpopulations (of total gated thymocytes). Sorting areas for “CD8-negative” (CD8-neg) and bona fide DP thymocytes for ChIP (Fig. 1b) and DNA methylation (Fig. 2) assays are indicated by rectangles. Data are representative of more than 10 mice analyzed. (b) Top: Map of the Cd8 gene loci after the deletion of E8I and E8II. Horizontal arrows indicate the transcriptional orientation of the Cd8 genes. Vertical arrows indicate the localization of DH sites that constitute clusters II, III, and IV39. The horizontal bars indicate genomic fragments used to define enhancers E8III, E8IV, and E8V as previously reported11, and Cd8a (P8a) and Cd8b1 (P8b) promoter regions. The triangles represent loxP sites left after the deletion of E8I and E8II. Only relevant BamHI (B) and EcoRI (E) sites are shown.

Article Snippet: The following antibodies were used for the stainings: PE-anti-mCD8α (CT-CD8a), TC-anti-mCD4 (CT-CD4), Cy5-anti-mCD3ε (500A2) and Cy5-anti-mCD8α (CT-CD8a) from Caltag, PE-anti-mCD5 (53-7.3) from Pharmingen.

Techniques: Expressing, Isolation, DNA Methylation Assay

Journal: Nature immunology

Article Title: Negative regulation of CD8 expression via CD8 enhancer-mediated recruitment of the zinc finger protein MAZR

doi: 10.1038/ni1311

Figure Lengend Snippet: DNA methylation of the promoter, exon, and intron regions of the Cd8a and Cd8b1 genes. Genomic DNA from DP and “CD8-negative” DP thymocytes was treated with sodium bisulfite and amplified by PCR as indicated by black thin lines (after ref. 23,24). Positions are indicated relative to the initation codon. Individual PCR products were cloned and sequenced, each line representing one clone. Four clones of each of three independently prepared thymocytes for each group were sequenced. Unmethylated CpG sites are indicated by open circles, whereas methylated CpG sites are indicated by filled circles.

Article Snippet: The following antibodies were used for the stainings: PE-anti-mCD8α (CT-CD8a), TC-anti-mCD4 (CT-CD4), Cy5-anti-mCD3ε (500A2) and Cy5-anti-mCD8α (CT-CD8a) from Caltag, PE-anti-mCD5 (53-7.3) from Pharmingen.

Techniques: DNA Methylation Assay, Amplification, Clone Assay, Methylation

Journal: Nature immunology

Article Title: Negative regulation of CD8 expression via CD8 enhancer-mediated recruitment of the zinc finger protein MAZR

doi: 10.1038/ni1311

Figure Lengend Snippet: Enhancer E8II binding properties and expression pattern of MAZR. (a) Electrophoretic mobility shift assay (EMSA) showing in vitro binding of MAZR or Myc-MAZR to E8II RE-1 (Supplementary Fig. 2b online). Open wedge indicates addition of increasing amounts of anti-Myc. Data are representative of two independent experiments. (b) Chromatin immunoprecipitation (ChIP) assay showing in vivo interaction of MAZR with RE-1 in DN thymocytes. Primer pairs used for PCR correspond to amplicon 6 (Supplementary Table 2 online). Input DNA was used undiluted, or at 1:3 and 1:10 dilutions. Data are representative of seven independent experiments (c) Real-time qPCR quantification of MAZR expression in various thymocyte subsets. MAZR (Zfp278) transcript abundance was normalized to that of Hprt1 transcripts. (d) Immunoblot analysis showing MAZR and N-CoR expression levels in sorted thymocyte subpopulations and peripheral splenic CD8+ and CD4+ T cells. Whole cell lysates from 3 × 106 cells were used for each population. Control, protein lysate generated from thymocytes that were transduced with MAZR retrovirus (see Methods). The arrow indicates the position of MAZR. Actin protein abundance was used to confirm equal loading. Data in c and d are representative of two independent experiments.

Article Snippet: The following antibodies were used for the stainings: PE-anti-mCD8α (CT-CD8a), TC-anti-mCD4 (CT-CD4), Cy5-anti-mCD3ε (500A2) and Cy5-anti-mCD8α (CT-CD8a) from Caltag, PE-anti-mCD5 (53-7.3) from Pharmingen.

Techniques: Binding Assay, Expressing, Electrophoretic Mobility Shift Assay, In Vitro, Chromatin Immunoprecipitation, In Vivo, Amplification, Western Blot, Generated, Transduction

Journal: Nature immunology

Article Title: Negative regulation of CD8 expression via CD8 enhancer-mediated recruitment of the zinc finger protein MAZR

doi: 10.1038/ni1311

Figure Lengend Snippet: Forced expression of MAZR in thymocytes induces variegation of CD8 expression. (a) Two-color flow cytometry of MAZR transduced wild-type thymocytes. Representative dot plots show CD4 versus CD8 expression. Numbers in the dot plot quadrants indicate the percentage of the corresponding thymocyte subpopulations of total gated GFP− (top) and GFP+ thymocytes (bottom), respectively. (b) Expression of CD3 and CD5 on CD4 SP (left), DP (middle) and CD8 SP (right) thymocytes (as defined by the gates in Fig. 4a). Top, expression of CD3 (left) and CD5 (right) in MIG-R-transduced thymocytes. Bottom, expression of CD3 (left) and CD5 (right) in MAZR-transduced thymocytes. Expression patterns for GFP+ and GFP− populations are indicated. Numbers in the CD4 SP-gated histograms indicate the percentage of cells within the indicated regions (i.e. CD3lo and CD5int, respectively) of total gated thymocytes. Data are representative of at least 5 experiments.

Article Snippet: The following antibodies were used for the stainings: PE-anti-mCD8α (CT-CD8a), TC-anti-mCD4 (CT-CD4), Cy5-anti-mCD3ε (500A2) and Cy5-anti-mCD8α (CT-CD8a) from Caltag, PE-anti-mCD5 (53-7.3) from Pharmingen.

Techniques: Expressing, Flow Cytometry

Journal: Nature immunology

Article Title: Negative regulation of CD8 expression via CD8 enhancer-mediated recruitment of the zinc finger protein MAZR

doi: 10.1038/ni1311

Figure Lengend Snippet: MAZR-induced variegation in wild-type, E8I- and E8II-deficient thymocytes (a) Representative GFP expression profile of thymocytes transduced with MIG-R and MAZR. Gating areas for GFP− (−) and GFP+ (+) cells (thin line rectangles), and high (hi) GFP-expressing cells (thick line rectangles) are shown. Scatter plots show the percentage of CD3lo (top) and CD5int (bottom) cells within the CD4 SP subset of MIG-R and MAZR transduced wild-type (wt; left), E8I-deficient (Δ1/Δ1; middle) or E8II-deficient (Δ2/Δ2; right) thymocytes, respectively. Each circle represents one mouse. Horizontal bars indicate average value. (b) Left, representative histograms of GFP+ thymocytes indicating gating areas for CD4+CD8-CD3lo, CD4+CD8-CD3hi and DP thymocytes, respectively, that were used to determine expression as shown in the right panel. Right, expression of the zinc finger transcription factor Th-POK in MAZR-trasnduced thymocytes. Semi-quantitative RT-PCR of Zbtb7b (Th-POK) mRNA levels in CD4+CD3lo, DP, and CD4+CD3hi populations of MAZR-transduced E8I-deficient thymocytes. Open wedges indicate undiluted sample, 1:3, and 1:10 dilutions, respectively. M, marker; −, RT(−) control. Hprt1 mRNA was used as quantification control.

Article Snippet: The following antibodies were used for the stainings: PE-anti-mCD8α (CT-CD8a), TC-anti-mCD4 (CT-CD4), Cy5-anti-mCD3ε (500A2) and Cy5-anti-mCD8α (CT-CD8a) from Caltag, PE-anti-mCD5 (53-7.3) from Pharmingen.

Techniques: Expressing, Transduction, Quantitative RT-PCR, Marker

Journal: Nature immunology

Article Title: Negative regulation of CD8 expression via CD8 enhancer-mediated recruitment of the zinc finger protein MAZR

doi: 10.1038/ni1311

Figure Lengend Snippet: MAZR is recruited to multiple sites within the Cd8a and Cd8b1 gene complex. (a) Top, schematic map of the Cd8a and Cd8b1 gene complex40. Horizontal arrows indicate the transcriptional orientation of the Cd8a and Cd8b1 genes. Vertical arrows indicate the localization of DH sites that constitute clusters II, III, and IV39. The horizontal thick bars below DH sites indicate genomic fragments used to define enhancers E8I, E8II, E8III, E8IV, and E8V as previously reported11, and Cd8a (P8a) and Cd8b1 (P8b) promoter regions. All BamHI (B) but only relevant EcoRI (E) sites are shown. Middle, PCR analysis of chromatin-immunoprecipitated DNA from sorted primary DN thymocytes. 14 amplicons spanning the Cd8 gene cluster are shown. Solid lines indicate PCR amplicons containing potential MAZR binding sites (1, 2, 3, 4, 5, 6, 8, 9, 10, 12 and 14), while dotted lines indicate control PCR amplicons (7, 11 and 13) within or outside of known enhancers that contain no potential MAZR binding sites. (b) Top, PCR analysis of chromatin-immunoprecipitated DNA from sorted primary DN and DP thymocytes. The 7 PCR amplicons (1, 2, 4, 6, 10, 12 and 14) showing MAZR binding in DN thymocytes (Fig. 6a) are shown. The input DNA in a and b was used undiluted, or at 1:3 and 1:10 dilutions to ensure PCR quantification within a non-saturated amplification range. Signal intensities at 1:3 input dilutions were used to calculate relative signal intensities for each amplicon. One representative out of three independent experiments is shown (Experiment 1 for a and experiment 3 for b). The relative densitometric quantification values (arbitrary units) of PCR signal intensities of precipitated DNA relative to the input fraction for each amplicon were calculated as described in the Methods section. The summary for three independent experiments is shown below. Boxed amplicons indicate results observed in all three ChIP experiments. (a) + : indicates binding; − : no binding. (b) > : indicates more MAZR binding in DN cells; < : less MAZR binding in DN cells; = : equal MAZR binding in DN and DP cells.

Article Snippet: The following antibodies were used for the stainings: PE-anti-mCD8α (CT-CD8a), TC-anti-mCD4 (CT-CD4), Cy5-anti-mCD3ε (500A2) and Cy5-anti-mCD8α (CT-CD8a) from Caltag, PE-anti-mCD5 (53-7.3) from Pharmingen.

Techniques: Immunoprecipitation, Binding Assay, Amplification

Journal: Nature immunology

Article Title: Negative regulation of CD8 expression via CD8 enhancer-mediated recruitment of the zinc finger protein MAZR

doi: 10.1038/ni1311

Figure Lengend Snippet: The BTB domain alone is not sufficient to induce variegated expression of CD8 in DP thymocytes. Scatter plot showing percentage of CD3lo cells within GFP−, GFP+, and GFPhi CD4 SP thymocytes of MIG-R, BTBMAZR, and MAZRΔZF transduced wild-type thymocytes. Representative gating areas for GFP expression are as in Fig. 5a. Each circle represents one mouse. Horizontal bars indicate average value.

Article Snippet: The following antibodies were used for the stainings: PE-anti-mCD8α (CT-CD8a), TC-anti-mCD4 (CT-CD4), Cy5-anti-mCD3ε (500A2) and Cy5-anti-mCD8α (CT-CD8a) from Caltag, PE-anti-mCD5 (53-7.3) from Pharmingen.

Techniques: Expressing