Fig. 3.

CXCL10 induces sustainable phosphorylation of p38 MAPK in CXCR3-positive T cells. (A) Histogram analysis of intracellular staining of p-p38 in CXCR3 positive lymphocytes (left) and mean fluorescent intensity of p-p38 in T cells (right). Freshly isolated human T cells were cultured in medium alone, or treated with IL-2, IFNα, or CXCL10 or a combination of the three, for the times indicated. Cells were then washed and analyzed by FACS. FACS was performed using an antibody specific for the activated p38 MAPK (phospho-p38 [p-p38]). The results are shown for events in the live cell gate and are representative of at least three independent experiments. (B) Western blots using a phospho-p38-specific antibody (left) were performed on protein derived from human T cells cultured either in medium alone or treated with CXCL10 + IL-2 + IFNα, for the times indicated. The phosphorylation was quantified by chemiluminescence and standardized against the same blots stripped and re-probed with antibodies that recognize total p38 protein. Actin was used as a loading control. Western blots and band quantification (right) in T cells that were untreated or cultured with IL-2, IFNα, CXCL10, or a combination of the three, as indicated, for 16 h. Cells were then lysed and phosphorylation of p38 was determined by western blot analysis using specific anti-phospho-p38 antibodies.