FIG. 6.

MEKK1 is increased during TCR stimulation while down-regulation of MEKK1 by siRNA inhibits T-cell activation. (A) TCR-mediated Deltex1 down-regulation was accompanied by an increase in MEKK1 protein levels. Splenic T cells were treated as described in the legend to Fig. 1D and MEKK1 contents were determined by immunoblotting. (B) TCR activation did not increase MEKK1 mRNA expression. Splenic T cells were stimulated as described in the legend to Fig. 1B and MEKK1 mRNA levels were assessed by reverse transcription-PCR. (C) Selective reduction of endogenous MEKK1 by siRNA. DO11.10 and EL4 cells were transfected with MEKK1-specific siRNA by electroporation with a Nucleofactor (Amaxa). The protein contents of MEKK1 were determined 48 h after transfection. (D and E) Decreased MEKK1 expression led to attenuated T-cell activation. EL4 (D) and DO11.10 cells (E), transfected with MEKK1-specific siRNA (solid column) or mock transfected (open column), were activated with plate-bound anti-CD3 and anti-CD28 48 h after transfection and IL-2 production was quantitated after another 16 h. (F) Down-regulation of MEKK1 impaired JNK activation. EL4 cells, transfected with MEKK1 siRNA or mock transfected, were stimulated through CD3/CD28, and JNK1 activity was determined as in Fig. 4A.