Suppression of PKD2 up-regulates Tyr307 phosphorylation of PP2A catalytic subunit after TCR stimulation.

<p>(A) Human CD4⁺ T cell clone was stimulated by L cells expressing its cognate ligands in the presence (closed circles) or absence (open circlse) of PKD2 inhibitor Gö6976 (3 µM). (B) Jurkat cells treated with siRNA were stimulated by anti-CD3ε antibody and anti-mouse IgG antibody. Treatment with siRNA2 (closed circles) significantly reduced the PKD2 expression, while siRNA1 (open circles) had almost no effect and hence used as negative controls (right panel). (C) Normal Jurkat cells (open circles) and Jurkat cells expressing GFP-SET-S171A (closed circles) were stimulated by anti-CD3ε antibody and anti-mouse IgG antibody. The latter cells expressed 4.5-fold higher GFP-SET-S171A than the endogenous SET as judged by the intensity of the bands using anti-SET antibody on the Western blot (upper box). Cell lysate were subjected to SDS-PAGE followed by western blot using anti-PP2A phospho-Tyr307 antibody and horseradish peroxidase-conjugated second antibody. The amount of each band was quantified by using ECL and LAS-4000. The same membrane was stripped and reprobed with the anti-PP2A C-subunit antibody to monitor relative abundance of total PP2A protein. Values represent the means ±SD from three independent experiments.