![Figure 6. CD3+ T-cell subset analysis shows a highly active PKA signaling node in CD8+CD45RO+ T cells. (A) Heatmap depicting differences in Arcsinh median of the antiphospho PKA substrate (RRXpS/pT) antibody in CD3+ T-cell subsets after 10 minutes of PGE2 (10μM) alone or with IBMX (3-isobutyl-1-methylxanthine) or rolipram (5mM and 20μM, respectively; 30 minutes) preincubation, or 8-CPT-cAMP (300μM; 5 minutes). Heatmap depicts net signaling profiles from 1 representative blood donor with the first column in each row set as reference. (B) Statistical analysis of PKA signaling in selected T-cell subsets from 3 individual blood donors analyzed independently as in panel A (mean ± SD; n = 3). (C) Heatmap depicting total signaling dynamics of the same samples as in panel A with the control sample of the CD3+ T-cell population set as reference. (D) Statistical analysis from 3 individual blood donors analyzed independently, using the difference in basal PKA signaling of C (mean ± SD; n = 3). (E) TCR (anti-CD3 [OKT3] 1 μg/mL; anti-CD28 [CD28.2] 5 μg/mL) stimulation for the indicated time period with and without PGE2 (10μM; 10 minutes preincubation) measured by Arcsinh median differences in CD3ζ-chain phosphorylation at Tyr142. Heatmap depicts the relative level of signaling with the control sample of the CD3+ T-cell population set as reference from 1 representative blood donor. (F) Statistical analysis from 5 individual blood donors analyzed independently (mean ± SD; n = 5) of samples as in panel E. To account for large differences in TCR responses between different donors, data are normalized to percentages with TCR stimulation (1 minute) representing 100%. (G-H) Heatmaps depicting total signaling dynamics for the indicated phospho-epitope–specific antibodies in CD8+ T cells from 1 representative blood donor with the control sample of the CD8+ T-cell population as reference. One minute of TCR (anti-CD3 [OKT3] 1 μg/mL; anti-CD28 [CD28.2] 5 μg/mL) stimulation and PGE2 (10μM) preincubation for 10 minutes before TCR stimulation. In all heatmaps, warmer colors (yellow) indicate increases and cooler colors (blue) indicate decreases in Arcsinh median differences displayed on a scale of 1.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/116/13/10.1182_blood-2010-01-266650/4/zh89991057590006.jpeg?Expires=1724224284&Signature=I~qmQkxArjru7FNc0Ii~GfppOJ4gbkMtl73KV6sGRbzgNGvPwgiQtTjN8QOQp3qzkLzOR6O6EKm2HN6T5WtrFsYe8POtqBXgaZTjI4VHzXIAcAU6oQ65GVFHALcycv07xYpow8lNeb3DMd6Ubhj4AFvaJqzzLphLtIaezPqxTHYja1GJZtYFTQ7kgMJNWJkAiTHb8wj1LnrA3u61Vgauv~0hx1Wier0S309ateEwh5wCXhnXrcPDZj6tCYnt83LQPUdGjMV9DG8NHhzAvZElD9QOCZzch6Ot~j2ho4bq-1kKBkNKDyUNrr2~knolJomAGTWqeAi4~IcDnuXeEhAtkA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
CD3+ T-cell subset analysis shows a highly active PKA signaling node in CD8+CD45RO+ T cells. (A) Heatmap depicting differences in Arcsinh median of the antiphospho PKA substrate (RRXpS/pT) antibody in CD3+ T-cell subsets after 10 minutes of PGE2 (10μM) alone or with IBMX (3-isobutyl-1-methylxanthine) or rolipram (5mM and 20μM, respectively; 30 minutes) preincubation, or 8-CPT-cAMP (300μM; 5 minutes). Heatmap depicts net signaling profiles from 1 representative blood donor with the first column in each row set as reference. (B) Statistical analysis of PKA signaling in selected T-cell subsets from 3 individual blood donors analyzed independently as in panel A (mean ± SD; n = 3). (C) Heatmap depicting total signaling dynamics of the same samples as in panel A with the control sample of the CD3+ T-cell population set as reference. (D) Statistical analysis from 3 individual blood donors analyzed independently, using the difference in basal PKA signaling of C (mean ± SD; n = 3). (E) TCR (anti-CD3 [OKT3] 1 μg/mL; anti-CD28 [CD28.2] 5 μg/mL) stimulation for the indicated time period with and without PGE2 (10μM; 10 minutes preincubation) measured by Arcsinh median differences in CD3ζ-chain phosphorylation at Tyr142. Heatmap depicts the relative level of signaling with the control sample of the CD3+ T-cell population set as reference from 1 representative blood donor. (F) Statistical analysis from 5 individual blood donors analyzed independently (mean ± SD; n = 5) of samples as in panel E. To account for large differences in TCR responses between different donors, data are normalized to percentages with TCR stimulation (1 minute) representing 100%. (G-H) Heatmaps depicting total signaling dynamics for the indicated phospho-epitope–specific antibodies in CD8+ T cells from 1 representative blood donor with the control sample of the CD8+ T-cell population as reference. One minute of TCR (anti-CD3 [OKT3] 1 μg/mL; anti-CD28 [CD28.2] 5 μg/mL) stimulation and PGE2 (10μM) preincubation for 10 minutes before TCR stimulation. In all heatmaps, warmer colors (yellow) indicate increases and cooler colors (blue) indicate decreases in Arcsinh median differences displayed on a scale of 1.
