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    Bethyl ps21 ezh2
    <t>Ezh2</t> is necessary for T FH lineage specification during the early activation stage. a Rapid induction of Ezh2 and Ser21 phosphorylation upon CD4 + T cell activation in vivo. CTV-labeled WT Smarta CD4 + T cells were adoptively transferred into B6.SJL recipients, either uninfected or infected with LCMV-Arm. Thirty-six hours later, undivided donor cells (Div0) or those in the first division (Div1) were analyzed for expression of CD25, total Ezh2 and <t>pS21-Ezh2.</t> Values denote geometric MFI, and data are representative from at least 2 independent experiments. b – c Predominant association of pS21-Ezh2 with nascent T FH cells at the fate-bifurcation stage. CTV-labeled Blimp1-YFP + Smarta CD4 + T cells were adoptively transferred into WT congenic recipients followed by LCMV infection. Sixty hours later, donor cells in the 5th division were analyzed for Blimp1-YFP in combination with surface staining of CXCR5 or CD25 or intracellular staining of T-bet and Tcf1 ( b ), or with total Ezh2 and pS21-Ezh2 ( c ). d – e Impact of Ezh2 deficiency on early T FH cells. CTV-labeled WT, Ezh2 –/– , or Ezh2 –/– Arf –/– Smarta CD4 + T cells were adoptively transferred, and recipients infected with LCMV-Arm as in ( b ). Cells in the 5th division at 60 h post-infection were analyzed for nascent T FH and T H 1 subsets based on CD25 and CXCR5 expression. The nascent T FH and T H 1 cells were then analyzed for expression of T H 1-associated Irf4 and T FH -associated Bcl6 ( d ), with values denoting geometric MFI and dotted lines marking histogram peaks in isotype control staining. Caspase-3/7 activation was determined in nascent T FH cells (lower panels in e ). Cumulative data are means ± s.d. from ≥2 independent experiments. ns, not statistically significant; * p
    Ps21 Ezh2, supplied by Bethyl, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Ezh2 is necessary for T FH lineage specification during the early activation stage. a Rapid induction of Ezh2 and Ser21 phosphorylation upon CD4 + T cell activation in vivo. CTV-labeled WT Smarta CD4 + T cells were adoptively transferred into B6.SJL recipients, either uninfected or infected with LCMV-Arm. Thirty-six hours later, undivided donor cells (Div0) or those in the first division (Div1) were analyzed for expression of CD25, total Ezh2 and pS21-Ezh2. Values denote geometric MFI, and data are representative from at least 2 independent experiments. b – c Predominant association of pS21-Ezh2 with nascent T FH cells at the fate-bifurcation stage. CTV-labeled Blimp1-YFP + Smarta CD4 + T cells were adoptively transferred into WT congenic recipients followed by LCMV infection. Sixty hours later, donor cells in the 5th division were analyzed for Blimp1-YFP in combination with surface staining of CXCR5 or CD25 or intracellular staining of T-bet and Tcf1 ( b ), or with total Ezh2 and pS21-Ezh2 ( c ). d – e Impact of Ezh2 deficiency on early T FH cells. CTV-labeled WT, Ezh2 –/– , or Ezh2 –/– Arf –/– Smarta CD4 + T cells were adoptively transferred, and recipients infected with LCMV-Arm as in ( b ). Cells in the 5th division at 60 h post-infection were analyzed for nascent T FH and T H 1 subsets based on CD25 and CXCR5 expression. The nascent T FH and T H 1 cells were then analyzed for expression of T H 1-associated Irf4 and T FH -associated Bcl6 ( d ), with values denoting geometric MFI and dotted lines marking histogram peaks in isotype control staining. Caspase-3/7 activation was determined in nascent T FH cells (lower panels in e ). Cumulative data are means ± s.d. from ≥2 independent experiments. ns, not statistically significant; * p

    Journal: Nature Communications

    Article Title: Ezh2 programs TFH differentiation by integrating phosphorylation-dependent activation of Bcl6 and polycomb-dependent repression of p19Arf

    doi: 10.1038/s41467-018-07853-z

    Figure Lengend Snippet: Ezh2 is necessary for T FH lineage specification during the early activation stage. a Rapid induction of Ezh2 and Ser21 phosphorylation upon CD4 + T cell activation in vivo. CTV-labeled WT Smarta CD4 + T cells were adoptively transferred into B6.SJL recipients, either uninfected or infected with LCMV-Arm. Thirty-six hours later, undivided donor cells (Div0) or those in the first division (Div1) were analyzed for expression of CD25, total Ezh2 and pS21-Ezh2. Values denote geometric MFI, and data are representative from at least 2 independent experiments. b – c Predominant association of pS21-Ezh2 with nascent T FH cells at the fate-bifurcation stage. CTV-labeled Blimp1-YFP + Smarta CD4 + T cells were adoptively transferred into WT congenic recipients followed by LCMV infection. Sixty hours later, donor cells in the 5th division were analyzed for Blimp1-YFP in combination with surface staining of CXCR5 or CD25 or intracellular staining of T-bet and Tcf1 ( b ), or with total Ezh2 and pS21-Ezh2 ( c ). d – e Impact of Ezh2 deficiency on early T FH cells. CTV-labeled WT, Ezh2 –/– , or Ezh2 –/– Arf –/– Smarta CD4 + T cells were adoptively transferred, and recipients infected with LCMV-Arm as in ( b ). Cells in the 5th division at 60 h post-infection were analyzed for nascent T FH and T H 1 subsets based on CD25 and CXCR5 expression. The nascent T FH and T H 1 cells were then analyzed for expression of T H 1-associated Irf4 and T FH -associated Bcl6 ( d ), with values denoting geometric MFI and dotted lines marking histogram peaks in isotype control staining. Caspase-3/7 activation was determined in nascent T FH cells (lower panels in e ). Cumulative data are means ± s.d. from ≥2 independent experiments. ns, not statistically significant; * p

    Article Snippet: For detection of pS21-Ezh2 (rabbit polyclonal, Bethyl Laboratories) or pT487-Ezh2 (rabbit polyclonal, abbexa, UK), the surface-stained and fixed cells were first stained with the primary antibody, followed by sequential staining with biotinylated goat anti-rabbit IgG (Cat. No. 111-066-144, Jackson ImmunoResearch Laboratories) and fluorochrome-conjugated streptavidin.

    Techniques: Activation Assay, In Vivo, Labeling, Infection, Expressing, Staining

    Ezh2 Ser21 phosphorylation is necessary for Bcl6 induction but dispensable for p19Arf repression. a Ezh2 phosphorylation status in polyclonal CD4 + T cell responses. CXCR5 + SLAM lo T FH cells and CXCR5 – SLAM hi T H 1 cells were sorted from activated CD4 + T cells in spleens of LCMV-Arm-infected WT C57BL/6 mice on 8 dpi, and immunoblotted with antibodies specific for pS21-Ezh2, pT487-Ezh2, and total Ezh2. Data are representative from 2 independent experiments with similar results. b Ezh2 phosphorylation status in monoclonal CD4 + T cell responses. CD45.2 + WT Smarta CD4 + T cells were adoptively transferred into congenic recipients followed by LCMV-Arm infection. On 4 dpi, CXCR5 + SLAM lo T FH cells and CXCR5 – SLAM hi T H 1 cells were analyzed for total Ezh2, pT487-Ezh2, or pS21-Ezh2 with intracellular staining. Values denote gMFI, and data are representative of ≥3 experiments. c – d Predominant association of pS21-Ezh2 with T FH lineage cells. Blimp1-YFP + Smarta CD4 + T cells were adoptively transferred into WT congenic recipients followed by LCMV infection. On 4 dpi, donor-derived CD4 + T cells were analyzed for Blimp1-YFP, CXCR5, T-bet, Tcf1 and pS21-Ezh2 at indicated combinations. e – g Effect of WT or mutant Ezh2 on rectifying T FH defects. Ezh2 –/– Smarta CD4 + T cells were primed in vitro and transduced with EV- GFP retrovirus or that expressing WT or mutant forms of Ezh2, followed by adoptive transfer and LCMV-Arm infection. WT Smarta CD4 + cells infected with EV- GFP retrovirus were used as a control. On 4 dpi, equivalent to day 7 after initial CD4 + T cell priming, GFP + Smarta CD4 + T cells were analyzed for frequency of CXCR5 + SLAM lo T FH cells ( e ). For a more accurate detection of the CXCR5 + Bcl6 + T FH subset in retrovirally transduced cells, CD45.2 + CD4 + T cells were intracellularly stained for Ezh2, and Ezh2 – cells in the EV- GFP group and Ezh2 + cells in other groups were analyzed ( f ). In ( g ), GFP + CD45.2 + CXCR5 + SLAM – T FH cells were sorted for analysis of Bcl6 , Arf , and Ink4a transcripts by quantitative RT-PCR. Cumulative data are means ± s.d. from ≥2 independent experiments. ns, not statistically significant; * p

    Journal: Nature Communications

    Article Title: Ezh2 programs TFH differentiation by integrating phosphorylation-dependent activation of Bcl6 and polycomb-dependent repression of p19Arf

    doi: 10.1038/s41467-018-07853-z

    Figure Lengend Snippet: Ezh2 Ser21 phosphorylation is necessary for Bcl6 induction but dispensable for p19Arf repression. a Ezh2 phosphorylation status in polyclonal CD4 + T cell responses. CXCR5 + SLAM lo T FH cells and CXCR5 – SLAM hi T H 1 cells were sorted from activated CD4 + T cells in spleens of LCMV-Arm-infected WT C57BL/6 mice on 8 dpi, and immunoblotted with antibodies specific for pS21-Ezh2, pT487-Ezh2, and total Ezh2. Data are representative from 2 independent experiments with similar results. b Ezh2 phosphorylation status in monoclonal CD4 + T cell responses. CD45.2 + WT Smarta CD4 + T cells were adoptively transferred into congenic recipients followed by LCMV-Arm infection. On 4 dpi, CXCR5 + SLAM lo T FH cells and CXCR5 – SLAM hi T H 1 cells were analyzed for total Ezh2, pT487-Ezh2, or pS21-Ezh2 with intracellular staining. Values denote gMFI, and data are representative of ≥3 experiments. c – d Predominant association of pS21-Ezh2 with T FH lineage cells. Blimp1-YFP + Smarta CD4 + T cells were adoptively transferred into WT congenic recipients followed by LCMV infection. On 4 dpi, donor-derived CD4 + T cells were analyzed for Blimp1-YFP, CXCR5, T-bet, Tcf1 and pS21-Ezh2 at indicated combinations. e – g Effect of WT or mutant Ezh2 on rectifying T FH defects. Ezh2 –/– Smarta CD4 + T cells were primed in vitro and transduced with EV- GFP retrovirus or that expressing WT or mutant forms of Ezh2, followed by adoptive transfer and LCMV-Arm infection. WT Smarta CD4 + cells infected with EV- GFP retrovirus were used as a control. On 4 dpi, equivalent to day 7 after initial CD4 + T cell priming, GFP + Smarta CD4 + T cells were analyzed for frequency of CXCR5 + SLAM lo T FH cells ( e ). For a more accurate detection of the CXCR5 + Bcl6 + T FH subset in retrovirally transduced cells, CD45.2 + CD4 + T cells were intracellularly stained for Ezh2, and Ezh2 – cells in the EV- GFP group and Ezh2 + cells in other groups were analyzed ( f ). In ( g ), GFP + CD45.2 + CXCR5 + SLAM – T FH cells were sorted for analysis of Bcl6 , Arf , and Ink4a transcripts by quantitative RT-PCR. Cumulative data are means ± s.d. from ≥2 independent experiments. ns, not statistically significant; * p

    Article Snippet: For detection of pS21-Ezh2 (rabbit polyclonal, Bethyl Laboratories) or pT487-Ezh2 (rabbit polyclonal, abbexa, UK), the surface-stained and fixed cells were first stained with the primary antibody, followed by sequential staining with biotinylated goat anti-rabbit IgG (Cat. No. 111-066-144, Jackson ImmunoResearch Laboratories) and fluorochrome-conjugated streptavidin.

    Techniques: Infection, Mouse Assay, Staining, Derivative Assay, Mutagenesis, In Vitro, Transduction, Expressing, Adoptive Transfer Assay, Quantitative RT-PCR