PKC-θ participates in the inducible expression of T-bet. (A) Calphostin C (0.05 μM) and rottlerin (30 μM) were used for the inhibition of PKC and PKC-θ, respectively. SB203580 (10 μM) and PD98059 (50 μM) were used for the inhibition of the p38 and the MEK/Erk kinase pathway, respectively. Normal T cells were treated with these inhibitors prior to stimulation with PMA and ionomycin followed by preparation of nuclear extracts. The nuclear extracts were used in immunoblot experiments for T-bet expression. In normal T cells, calphostin did not have any effect on T-bet expression, while rottlerin inhibited T-bet protein levels by 50%. SB203580 and PD98059 decreased T-bet levels by 35% and 10%, respectively. The results shown here are representative of 5 different experiments performed. (B) When T cells from aplastic anemia patients were treated with rottlerin, T-bet expression was decreased by 50%. (C) SB203580 showed a mean 15% decrease in T-bet protein levels in aplastic anemia patients.