PE anti-mouse PLZF 145803 from BioLegend

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eLife
April 05 2022

Fig 1: Characterization of m-reMAIT cells.(A) Flow cytometric profiles of m-reMAIT cells. mMR1-tet staining and expression of T-cell receptor ß (TCRß), CD4, CD8, CD25, and CD44 and the transcription factors PLZF and ROR?t in m-reMAIT cells on differentiation day 18. (B) 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) dose-dependent activation of m-reMAIT cells. The percentages of CD69+ cells among m-reMAIT cells challenged with the indicated concentration of 5-OP-RU in the presence of WT3 (?), WT3/mMR1 (?), CH27 (?), and CH27/mMR1(¦). (C) MR1-dependent activation of m-reMAIT cells. The percentage of CD69+ cells among m-reMAIT cells cultured with CH27/mMR1, challenged as in (B) in the presence of the anti-MR1 antibody (¦) or the isotype control antibody (?). (D) 5-OP-RU dose-dependent activation. The percentage of m-reMAIT cells expressing CD69 upon a challenge with various concentrations of 5-OP-RU. Representative data from two independent experiments are shown. (E) mMR1-tet dose-dependent activation. The percentage of m-reMAIT cells expressing CD69 upon a challenge with the indicated amounts of mMR1-tet. Representative data from two independent experiments are shown. (F) 5-OP-RU- and mMR1-tet-induced cytokines and chemokines. m-reMAIT cells were stimulated with various concentrations of 5-OP-RU (?) or mMR1-tet (¦) and the resultant cytokines and chemokines were quantified with LegendPlex. The concentrations at which each reagent induced a similar degree of activation (% CD69) are shown as relative concentrations (0.1–100 nM for 5-OP-RU and 0.01–10 µg/ml for mMR1-tet). The number on the X-axis corresponds to that in (D) and (E). (G) Tyrosine phosphorylation elicited with 5-OP-RU. A Western blot analysis with PY99 (anti-phosphotyrosine). Upon a challenge with different concentrations of 5-OP-RU for 30 min, the cell lysate from m-reMAIT cells was separated on SDS-PAGE (5 × 105/lane), and subjected to Western blotting. Lane 1, 0; lane 2, 0.1; lane 3, 1.0; lane 4, 10; lane 5, 100; lane 6, 1000; and lane 7, 10,000 (nM). Phosphorylated proteins are indicated with arrows. (H) Time course of tyrosine phosphorylation. A Western blot analysis with PY99. The cell lysate from m-reMAIT cells challenged with 100 nM of 5-OP-RU for the indicated time was separated on SDS-PAGE (5 × 105/lane) and subjected to Western blotting. Lane 1, 0; lane 2, 15; lane 3, 30; lane 4, 60; lane 5, 150; lane 6, 300 (min) . Arrows indicate phosphorylated proteins. (I) Linker for the activation of T cells (LAT) as a phosphorylated 37-kD protein. A Western blot analysis with PY99, anti-LAT, and anti-ß-actin. The cell lysate prepared as described in (H) for 60 min was subjected to Western blotting. A blot with PY99 (upper panel), anti-LAT (middle panel), and anti-ß-actin (lower panel). Phosphorylated LAT and LAT as well as ß-actin are indicated (arrow). Lane 1, 0; lane 2, 0.1; lane 3, 1.0; lane 4, 10; lane 5, 100; lane 6, 1000; lane 7, 10,000 (nM). (J) Tyrosine phosphorylation induced by mMR1-tet. A Western blot analysis with PY99. The cell lysate from m-reMAIT cells challenged with the indicated amounts of unlabeled mMR1-tet for 60 min was subjected to Western blotting (5 × 105/lane). Lane 1, 0; lane 2, 0.43; lane 3, 1.3; lane 4, 4.3; lane 5, 13 (µg/ml). Arrows indicate phosphorylated proteins. MAIT: mucosal-associated invariant T cell. Figure 1—source data 1.Characterization of m-reMAIT cells.The type of antigen-presenting cell (APC), reMAIT cells, and the concentration of 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) (nM) used for activation assays. Activation is shown as the percentage of CD69+ cells among reMAIT cells (B). The percentage of CD69+ MAIT cells challenged with 5-OP-RU in the presence of isotype control or anti-MR1 antibody (in the presence of CH27m as APC) (C). The percentage of CD69+ MAIT cells challenged with 5-OP-RU or mMR1-tet (D, E). Production of the cytokines and chemokines from m-reMAIT cells challenged with 5-OP-RU or mMR1-tet (F). MAIT: mucosal-associated invariant T cell. Figure 1—source data 2.Original gel electrophoresis panels for Figure 1G–J.
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iScience
March 17 2023

Fig 2: γδ thymocytes from Rag1Cre-Nfatc1fl/fl mice exhibit an NKT γδ phenotype(A) Differently expressed genes from freshly isolated Rag1Cre-Nfatc1fl/fl and Nfatc1fl/fl γδ thymocytes.(B) Cell type identification analysis of RNA-seq data of Nfatc1fl/fl and RagCre-Nfatc1fl/fl γδ thymocytes shows the dominance of NKT cell phenotype.(C) Flow cytometry of γδ thymocytes from Nfatc1fl/fl and RagCre-Nfatc1fl/fl mice upon staining with anti-CD4 and anti-CD8. Right, percentages of CD4+ γδ thymocytes.(D) Flow cytometry of thymocytes from Nfatc1fl/fl and RagCre-Nfatc1fl/fl mice gated for CD4+ TCRδ+ cells upon staining with anti-NK1.1. Right, percentages of CD4+ TCRδ+ thymocytes expressing the NK1.1 surface marker.(E) Flow cytometry of total thymocytes from Nfatc1fl/fland RagCre-Nfatc1fl/fl mice gated for CD4+ TCRδ+ cells upon staining with anti-Vγ1.1 and anti-Vδ6.3. Right, percentages of CD4+ TCRδ+ thymocytes expressing the Vγ1.1+ and Vδ6.3+ gene segments.(F) Number of γδ thymocytes from Nfatc1fl/fl and RagCre-Nfatc1fl/fl mice expressing the transcription factor PLZF analyzed by flow cytometry (left) and percentages of γδ thymocytes expressing PLZF (right).(G) (Above) Flow cytometry analysis of PLZF+ γδ thymocytes from WT (CD45.1+CD45.2+) and Rag1Cre-Nfatc1f/fl (CD45.2+) mouse bone marrow chimera. Total thymocytes are gated for TCRδ and TCRβ expression. The percentages of γδ PLZF+ thymocytes is calculated from the WT and the Rag1Cre-Nfatc1f/fl γδ thymocyte compartments. Data are representative of at least two independent experiments with four mice from every genotype and are shown as mean ± SEM. The statistical significance was determined by unpaired student’s t-tests. ∗∗p value <0.005, ∗∗∗p value <0.001, ∗∗∗∗p value <0.000. See also Figure S2.
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iScience
March 17 2023

Fig 3: The absence of NFATc1 confers a better survival rate to PLZF+ TCRδ6.3+ thymocytes from Rag1Cre-Nfatc1fl/fl mice(A) Gene Ontology (GO) analysis of the genes differentially expressed in Rag1Cre-Nfatc1fl/fl compared to control thymocytes. The red star indicates the enrichment of genes involved in “positive regulation of cell death” pathway downregulated in NFATc1-deficient γδ thymocytes.(B) Annexin V (left), Bcl2 (mid), and ki67 (right) staining of Rag1Cre-Nfatc1fl/fl and control thymocytes gated for the expression of PLZF and TCRVδ6.3 gene segment.(C) Corresponding percentages and absolute cell numbers of PLZF+ TCRVδ6.3- and TCRVδ6.3+ thymocyte populations analyzed by flow cytometry as reported in (b). (Left) percentages and absolute cell numbers of PLZF+ TCRVδ6.3- and TCRVδ6.3+ subpopulations, and (right) percentages of annexin V+ and absolute numbers of annexin V+ and annexin V− PLZF+ TCRVδ6.3- and TCRVδ6.3+ thymocytes.(D) Percentages of Bcl2-expressing PLZF+ TCRVδ6.3- and TCRVδ6.3+ thymocytes (left) and percentages of PLZF+ TCRVδ6.3- and TCRVδ6.3+ positively stained for ki67 protein (right).(E) Cell numbers of Rag1Cre-Nfatc1fl/fl and control γδ thymocytes expressing PLZF stimulated with αCD3, or αCD3 and CsA for 24 h or left unstimulated. (Right) Percentages of γδ thymocytes expressing PLZF. Data are representative of at least two independent experiments with four mice from every genotype and are shown as mean ± SEM. The statistical significance was determined by unpaired student’s t-tests. ∗p value <0.05, ∗∗p value <0.005, ∗∗∗p value <0.001, n.s. not significant. See also Figure S3.

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PE anti-mouse PLZF

The PE anti-mouse PLZF Antibody from BioLegend is a Hamster Monoclonal antibody to ZBTB16. This antibody recognizes Mouse antigen. The PE anti-mouse PLZF Antibody has been shown to work in the following applications: Flow Cytometry.

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PE anti-mouse PLZF [9E12];

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PE anti-mouse PLZF from BioLegend

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PE anti-mouse PLZF from BioLegend

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