Figure 3.

Activity in T cells. (a) Jurkat T cells were resuspended in growth medium and stimulated with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml), anti-CD3 (0.5 μg/ml), and anti-CD28 (2 μg/ml) for 30 min. SP600125 was added as a 10-min pretreatment at the concentrations indicated. The same cell lysates were examined by using different Abs targeting mitogen-activated protein kinase and NF-κB signaling pathways. Levels of nonphosphorylated IKK-1 were used as an indication of equal loading. (b) Inhibition of CD4⁺ cell activation and differentiation. Th0 cells isolated from either human cord or peripheral blood were cultured with anti-CD3/antiCD28, IL-12, anti-IL-4 in the absence or presence of SP600125 (30 μM). Culture aliquots were analyzed at different times for the expression of cell surface markers CD4, CD45RO, CXCR4, and CCR5. (c) CD4⁺ cells were differentiated into either Th1 or Th2 cultures over 5 days by using polarizing conditions before stimulating with anti-CD3/anti-CD28 in the presence of increasing concentrations of SP600125 for 15 h in triplicate. Culture supernatants were analyzed by simultaneous analyte reagent technology multiplex cytokine bead array. Data shown represent one of the duplicate experiments. Results are expressed as the mean of triplicate samples.