superscript iii reverse transcriptase kit  (Meridian Bioscience)

Figure Legend Snippet: ( a – c ) Naive CD4 + T cells were activated by anti-CD3 and anti-CD28. At indicated time points after activation, cell growth was analysed by microscopy for morphology ( a ), by bicinchoninic acid assay (BCA) assay for protein amount ( b ), and by flow-cytometry for cell size ( c ). Means±s.d. of three experiments are shown (* P <0.05 by Student's t- test; NS, not significant, P >0.05 by Student's t -test). Representative results of at least three independent experiments are shown. ( d , e ) The amount of DNA synthesis was determined by BrdU incorporation ( d ), and the proliferation was assessed by CFSE dilution ( e ) of CD4 + naive T cells activated by anti-CD3 and anti-CD28 at indicated time points. Results are representative of three experiments. ( f , g ) The expression of DCAF1 was monitored by immunoblotting ( f ) and quantitative reverse transcription–PCR ( g ) assays at indicated time points after CD4 + naive T cells were activated by anti-CD3 and anti-CD28. Results are representative of three experiments.

Techniques Used: Activation Assay, Microscopy, Acid Assay, BIA-KA, Flow Cytometry, DNA Synthesis, BrdU Incorporation Assay, Expressing, Western Blot

Figure Legend Snippet: ( 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 <0.05 by Student's t- test). See also .

Techniques Used: Expressing, Activation Assay, Western Blot, DNA Synthesis, BrdU Incorporation Assay

Figure Legend Snippet: ( 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 .

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