Figure 3.

CXCR3 agonists and CXCL4 induce phosphorylation of p44/p42 extracellular signal-regulated kinase (ERK) and protein kinase B (PKB)/Akt. Aliquots of activated T cells (1 × 10⁶ cells/500 μl) were left untreated or stimulated in parallel at 37° with 0.3–100 nm CXCR3 agonist for 2 min. Cells were lysed by the addition of 1 × sample buffer. Cell lysates were resolved by SDS–PAGE, transferred to nitrocellulose membranes, and immunoblotted with a phospho-specific p44/42 ERK or Akt antibody with affinity for the active Ser473-phosphorylated form of Akt and proteins were visualized with enhanced chemiluminescence. The blots were stripped and reprobed with anti-Erk1 antibody to verify equal loading and efficiency of protein transfer (lower panel). p44/p42 ERK and Akt phosphorylation (mean from four experiments, ±SEM) was quantified by densitometry and corrected for total Erk1 expression on stripped blots. The immnoblots shown are derived from a single experiment representative of at least four others using cells from different donors.