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Downregulation of p53 is required for DCAF1-dependent cell cycle entry. ( a – d ) CD4 + naive T cells of different genotypes were activated by anti-CD3 and anti-CD28 at indicated time points. The cell size measured by flow-cytometry ( a ), the cell proliferation determined by CFSE dilution assay ( b ), the amounts of <t>DNA</t> synthesis measured by BrdU incorporation assay 24 h post activation ( c ), and p53 and c-Myc protein expression assessed by immunoblotting ( d ) were compared. ( e ) Comparison of the proliferation of CD4 + naive T cells of indicated genotypes (lines) to that of wild-type CD4 + T cells (shaded area) determined by CFSE dilution assay at indicated time points post anti-CD3 and anti-CD28 activation in the presence of 4-hydroxy-tamoxifen. ( f , g ) CD4 + naive T cells of different genotypes were activated by anti-CD3 and anti-CD28 for 5 days to generate effector T cells in the presence of 4-hydroxy-tamoxifen. Quiescent effector T cells were either re-stimulated with IL-2 for 24 h or remained unstimulated (quiescence). The amounts of DNA synthesis were determined by BrdU incorporation assay ( f ). The protein expression of p53 and c-Myc was analysed by immunoblotting ( g ). In this figure, representative flow-cytometry and immunoblotting results of <t>three</t> experiments are shown. For BrdU incorporation assay results ( c , f ), means±s.d. of three experiments are shown. See also Supplementary Fig. 6 .
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Article Title: DCAF1 controls T-cell function via p53-dependent and -independent mechanisms

Journal: Nature Communications

doi: 10.1038/ncomms10307

Downregulation of p53 is required for DCAF1-dependent cell cycle entry. ( a – d ) CD4 + naive T cells of different genotypes were activated by anti-CD3 and anti-CD28 at indicated time points. The cell size measured by flow-cytometry ( a ), the cell proliferation determined by CFSE dilution assay ( b ), the amounts of DNA synthesis measured by BrdU incorporation assay 24 h post activation ( c ), and p53 and c-Myc protein expression assessed by immunoblotting ( d ) were compared. ( e ) Comparison of the proliferation of CD4 + naive T cells of indicated genotypes (lines) to that of wild-type CD4 + T cells (shaded area) determined by CFSE dilution assay at indicated time points post anti-CD3 and anti-CD28 activation in the presence of 4-hydroxy-tamoxifen. ( f , g ) CD4 + naive T cells of different genotypes were activated by anti-CD3 and anti-CD28 for 5 days to generate effector T cells in the presence of 4-hydroxy-tamoxifen. Quiescent effector T cells were either re-stimulated with IL-2 for 24 h or remained unstimulated (quiescence). The amounts of DNA synthesis were determined by BrdU incorporation assay ( f ). The protein expression of p53 and c-Myc was analysed by immunoblotting ( g ). In this figure, representative flow-cytometry and immunoblotting results of three experiments are shown. For BrdU incorporation assay results ( c , f ), means±s.d. of three experiments are shown. See also Supplementary Fig. 6 .
Figure Legend Snippet: Downregulation of p53 is required for DCAF1-dependent cell cycle entry. ( a – d ) CD4 + naive T cells of different genotypes were activated by anti-CD3 and anti-CD28 at indicated time points. The cell size measured by flow-cytometry ( a ), the cell proliferation determined by CFSE dilution assay ( b ), the amounts of DNA synthesis measured by BrdU incorporation assay 24 h post activation ( c ), and p53 and c-Myc protein expression assessed by immunoblotting ( d ) were compared. ( e ) Comparison of the proliferation of CD4 + naive T cells of indicated genotypes (lines) to that of wild-type CD4 + T cells (shaded area) determined by CFSE dilution assay at indicated time points post anti-CD3 and anti-CD28 activation in the presence of 4-hydroxy-tamoxifen. ( f , g ) CD4 + naive T cells of different genotypes were activated by anti-CD3 and anti-CD28 for 5 days to generate effector T cells in the presence of 4-hydroxy-tamoxifen. Quiescent effector T cells were either re-stimulated with IL-2 for 24 h or remained unstimulated (quiescence). The amounts of DNA synthesis were determined by BrdU incorporation assay ( f ). The protein expression of p53 and c-Myc was analysed by immunoblotting ( g ). In this figure, representative flow-cytometry and immunoblotting results of three experiments are shown. For BrdU incorporation assay results ( c , f ), means±s.d. of three experiments are shown. See also Supplementary Fig. 6 .

Techniques Used: Flow Cytometry, Cytometry, Dilution Assay, DNA Synthesis, BrdU Incorporation Assay, Activation Assay, Expressing

DCAF1 is essential for cell cycle entry. ( a ) DCAF1 protein expression in CD4 + T cells of different genotypes at indicated time points after TCR activation and 4-hydroxy-tamoxifen treatment, analysed by immunoblotting. The immunoblotting is representative of at least three experiments. ( b – d ) Equal numbers of wild-type (CD45.1 + ) and ERCre ; Dcaf1 fl/fl (CD45.2 + ) CD4 + T cells were mixed and activated with anti-CD3 and anti-CD28 in the presence of 4-hydroxy-tamoxifen. At indicated time points after activation, the cell sizes ( b ), the proliferation ( c ) and the amount of DNA synthesis (measured by BrdU incorporation assay) ( d ) of the T cells of different genotypes were assessed and compared. Results are representative of at least three experiments. ( e , f ) CD4 + T cells from wild-type (CD45.1 + ) and ERCre ; Dcaf1 fl/fl (CD45.2 + ) mice were mixed and activated by anti-CD3 and anti-CD28 in the presence of 4-hydroxy-tamoxfin for 5 days for them to become effector T cells. Quiescent effector T cells were either re-stimulated with IL-2 or remained unstimulated (quiescence). The amount of DNA synthesis (measured by BrdU incorporation assay) ( e ) and the sizes ( f ) of the cells of different origins were compared. The bar graphs show the means±s.d. of data from four experiments (* P
Figure Legend Snippet: DCAF1 is essential for cell cycle entry. ( a ) DCAF1 protein expression in CD4 + T cells of different genotypes at indicated time points after TCR activation and 4-hydroxy-tamoxifen treatment, analysed by immunoblotting. The immunoblotting is representative of at least three experiments. ( b – d ) Equal numbers of wild-type (CD45.1 + ) and ERCre ; Dcaf1 fl/fl (CD45.2 + ) CD4 + T cells were mixed and activated with anti-CD3 and anti-CD28 in the presence of 4-hydroxy-tamoxifen. At indicated time points after activation, the cell sizes ( b ), the proliferation ( c ) and the amount of DNA synthesis (measured by BrdU incorporation assay) ( d ) of the T cells of different genotypes were assessed and compared. Results are representative of at least three experiments. ( e , f ) CD4 + T cells from wild-type (CD45.1 + ) and ERCre ; Dcaf1 fl/fl (CD45.2 + ) mice were mixed and activated by anti-CD3 and anti-CD28 in the presence of 4-hydroxy-tamoxfin for 5 days for them to become effector T cells. Quiescent effector T cells were either re-stimulated with IL-2 or remained unstimulated (quiescence). The amount of DNA synthesis (measured by BrdU incorporation assay) ( e ) and the sizes ( f ) of the cells of different origins were compared. The bar graphs show the means±s.d. of data from four experiments (* P

Techniques Used: Expressing, Activation Assay, DNA Synthesis, BrdU Incorporation Assay, Mouse Assay

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Real-time Polymerase Chain Reaction:

Article Title: DCAF1 controls T-cell function via p53-dependent and -independent mechanisms
Article Snippet: .. RNA preparation and real-time PCR Total RNA was prepared from T cells using TRIzol reagent (Invitrogen, 15596-026) as per manufacturer's instructions, and was reverse-transcribed into complementary DNA with Superscript III reverse transcriptase kit (Bioline, BIO-65054). .. The Taqman probes of p21 (Mm04205640_g1), c-Myc (Mm00487804), Bax (Mm00432051), Dcaf1 (Mm01226827_g1) and Hprt (Mm01545399) were from Applied Biosystems and Quantitative PCR was performed using SensiFastTM Probe Lo-ROX Kit (Bioline, BIO-84020) on ABI9700 real-time PCR system.