Knockdown of NFATc1 and NFATc3 has differential effects on induced expression of IL2 and COX2 in Jurkat cells. (A) Representative Western blot showing expression of NFATc1, NFATc2, and NFATc3 in total extracts of resting Jurkat cells or cells activated with PMA + Io. (B) HEK cells were transfected with pcDNA3.1 NFATc1 or c3 alone (−) or together with pSupeRetro shRNA sequences targeting NFATc1 (KDc1) and NFATc3 (KDc3). KDc1 shRNA #3 and KDc3 shRNA #2 were selected. (C) Jurkat cells were infected with the indicated knock-down LV vectors: Sc (control), c1 (NFATc1), and c3 (NFATc3). Expression of NFATc1, NFATc2, and NFATc3 protein was detected in lentivirus-infected Jurkat cells stimulated with PMA + Io for 5 hours. PTB-associated splicing Factor expression was monitored as a loading control for NFATc1 and NFATc3; tubulin expression was monitored as a loading control for NFATc2. (D) IL2 mRNA expression determined by qPCR after 2 hours of stimulation. (E) IL2 release measured by ELISA after 8 hours of stimulation. (F) COX2 mRNA levels determined by qPCR after 2 hours of stimulation. (G) Representative Western blot showing COX2 protein expression after 24 hours of stimulation. Tubulin (TUB) was used as a loading control. *P < .05, **P < .01, and ***P < .001 compared with control (KDSc) (n = 3).