A). Diagram showing sequence alignment and relative location of the X-H region in the 3’UTR of human and murine CD154 mRNA. B). Human (lanes 1–5) and murine (lanes 6–10) X-H region probes labeled with alpha-³²P-UTP were used in binding studies with cytoplasmic extracts from human Jurkat/D1.1 cells. Anti-PTB (lanes 5 and 10) or isotype control (lanes 4 and 9) antibodies were included to identify PTB-containing complexes (denoted by asterisk). Lanes 1 and 6 show probes alone and reactions in lanes 2 and 7 contained probe plus RNase without extract. Closed and open arrows designate human Complex I and II, respectively, whereas the square bracket indicate the broad complex binding to the mouse X-H region. C). Cytoplasmic extracts were prepared from splenic CD4+ T cells (lanes 3–8) that were either resting (lanes 3, 5 and 7) or activated with anti-CD3 and anti-CD28 mAbs for 24 h (lanes 4, 6 and 8). RNA-EMSA was carried out using the mouse X-H region probe labeled with alpha-³²P-UTP. Comparison reactions were established using Jurkat/D1.1 cytoplasmic extracts (lanes 9–11). Anti-PTB (lanes 7, 8 and 11) and control isotype (lanes 5, 6 and 10) antibodies were added to specific reactions to establish the presence of PTB. The open and closed arrows indicate the PTB-containing complexes in resting and activated CD4+ T cells, respectively.

A). Changes in number, ratio and phenotype of splenocytes detected by FACS analysis are recorded over time (6 and 24 h) following injection of anti-CD3 and anti-CD28 mAbs compared to injection of isotype control antibodies. This data reflects the average and SEM of 3 independent experiments. B). The t_1/2 of CD154 mRNA in splenocytes isolated from mice injected with anti-CD3 and anti-CD28 mAbs or isotype control antibodies for 6 and 24 h was measured by real-time PCR following incubation with the transcription inhibitor DRB for periods of 0, 20, 40 and 60 min. This data represent the average and +/− SEM (error bars) of 3 independent experiments.

A). Shown is representative of 3 flow cytometric analyses of CD154 surface expression using a PE-conjugated mAb with purified spleenic CD4+ T cells activated for 0, 6, 12, 24 and 48 h with anti-CD3 and anti-CD28 mAbs. Dotted line indicates isotype-PE control. B). Steady-state expression of CD154 mRNA was determined in samples after stimulation with anti-CD3 and anti-CD28 mAbs for indicated times. Fold differences of CD154 mRNA molecules relative to resting cells were normalized using β-actin mRNA and results are the average +/− SEM of 3 independent experiments. C). CD4⁺ T cells were stimulated for 0, 6, 12, 24 and 48 h with anti-CD3 and anti-CD28 mAbs followed by treatment with DRB for 15, 30, 45 and 60 min. RNA was isolated and CD154 expression analyzed by real-time PCR to determine the t_1/2 of the CD154 transcript. Results were normalized using β-actin transcripts and plotted as the fraction of CD154 mRNA remaining relative to time 0 for each DRB time point. These results represent the average +/− SEM of 3 independent experiments. In resting (0 h) no CD154 mRNA was detectable beyond 30 min of DRB treatment.

A). Diagram showing the relative location of sequence features within the mouse X-H region and the sequence location of probes A, B and C. B). Cytoplasmic extracts were prepared from splenic CD4+ T cells (lanes 9–28) that were either resting (lanes 9–12 and 21–24) or activated with anti-CD3 and anti-CD28 mAbs for 6 h (lanes 13–16) or 24 h (lanes 17–20 and 25–28). RNA-EMSA was carried out using the mouse X-H probe (lanes 9, 13, 17, 21 and 25) and probes A (lanes 10, 14, 18, 22 and 26), B (lanes 11, 15, 19, 23 and 27) and C (lanes 12, 16, 20, 24 and 28) labeled with alpha-³²P-CTP. Anti-PTB (lanes 21–28) and anti-nucleolin (lanes 29–32) mAbs were added to reactions to super-shift complexes (filled arrow). mComplex I is indicated by middle bracket (mCI) and an upper (u) and lower (l) minor complexes are indicated. C). Alpha-³²P-UTP-labeled RNA probes corresponding to the mouse X-H, A, B and C regions were incubated with cytoplasmic extracts from resting (lanes 9–12) or 24 h activated (lanes 13–16) mouse CD4+ T cells or human Jurkat/D1.1 cells (lanes 17–20) followed by RNAse treatment. The migration of the PTB-mRNA complex is shown with a bracket while filled and empty arrows point to slow and fast migrating ribonucleoproteins, respectively. The same sets of probes were incubated with cytoplasmic extracts from 24 h activated mouse CD4+ T cells and the mRNA-PTB complex immunoprecipitated (PTB-IP) using PTB mAb (lanes 21 to 24). The same reaction was repeated using isotype control, which did not yield any band (data not shown). Intact probes are shown in lanes 1–4, while RNase treated probes in the absence of extract are shown in lanes 5–8.

A). CD69 expression in cells suspensions of draining lymph nodes of mice primed with either CFA alone (left graph grey, filled peak) or KLH + CFA (left graph, black, open peak). The same analysis was carried out following ex vivo incubations of primed cells with KLH for 24 h (right graph). This data is representative of three independent experiments. B). Analysis of CD154 mRNA decay in cells isolated from mice that were primed 9 days earlier with either CFA alone (open circle) or KLH + CFA (closed square) (left graph). The same samples were incubated ex vivo with KLH for 24 h (right graph) and real-time PCR was carried out on both samples following incubation with DRB for periods of 0, 20, 40 and 60 min. This data represents the average and +/− SEM of 3 independent experiments.