FIG. 4.

Deltex interferes with activation of JNK, ERK, and p38 MAPK but not of LAT and PKCθ. (A) Inhibition of CD3/CD28-simulated JNK activation by Deltex1. EL4 T cells, transfected with pcDNA4 or pcDNA4-Deltex, were stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (2.5 μg/ml) and total cellular extracts were prepared at the indicated time points. JNK1 was then immunoprecipitated from cellular extracts (100 μg), and the kinase activity of the immune complexes was determined using GST-c-Jun (1-79) as substrate. The amount of JNK1 in 20% of the immune complexes was determined by immunoblotting. (B and C) Deltex interfered with p42/p44 ERK activation and p38 MAPK stimulation. ERK and p38 activation in DO11.10 T cells before and after CD3/CD28 (5 versus 2.5 μg/ml) stimulation was determined using anti-phosphorylated T202/Y204 ERK and anti-phospho-T180/Y182 p38 MAPK, respectively. The levels of ERK2 and p38α MAPK were used as an internal control. (D) Deltex did not affect TCR-mediated LAT and PKCθ activation. LAT and PKCθ activation in YFP- or Deltex1-DO11.10 T cells were determined using anti-phospho-LAT (Y191) and anti-phospho-PKCθ (T538). The protein level of β-tubulin was used as an internal control.