Rapid induction of Ser175 phosphorylation of CDK9 after stimulation of resting memory CD4+ T cells through the TCR.

Abstract

<p>(A) Flow cytometry. Memory CD4⁺ T-cells isolated from healthy donors were stimulated for 2 hr or 24 hr with α-CD3 and α-CD28 mAbs to activate the TCR, immunostained with the flourophore-conjugated antibodies against total CDK9, pThr186 CDK9, CycT1 and pSer175 CDK9 and analyzed by multicolor flow cytometry. Top panels: Representative data from Experiment 1 (<b>Figs. S5 and S6</b>). Bottom panels: Representative data from Experiment 2 (<b>Figs. S7 and S8</b>). (B) Kinetic analysis. Time course of P-TEFb activation in primary resting memory CD4⁺ T-cells stimulated between 0 and 24 hrs with α-CD3 and α-CD28 mAbs or with PMA. Kinetic data from two TCR and one PMA activation (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003338#ppat.1003338.s009" target="_blank">Figs. S9</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003338#ppat.1003338.s010" target="_blank">S10</a></b>) experiments are shown. The fraction of positive cells staining for pThr186 (blue lines), CycT1 (red lines), pSer175 CDK9 (black lines), and total CDK9 (black lines) were measured by flow cytometry as shown in Panel A. Note that during the course of the experiment there is also a 2- to 10-fold increase in the mean fluorescent intensity for the CDK9 and CycT1 proteins that is not represented by the data for % positive cells.</p