Proinflammatory cytokine production is inhibited by dasatinib. (A) PBMCs were stimulated with OKT3 (100 ng/mL) in the presence or absence of dasatinib (10 nM). After 24 hours, supernatants (Sn) were collected and T cells were analyzed for CD69 expression, as in Figure 3. Cytokines in the supernatants were detected by capture antibodies spotted in duplicate on nitrocellulose membranes (R&D Systems). Chemiluminescence signal was detected with a FluorChem SP CCD camera and intensity was quantitated using Quantity One 1-D Analysis Software (Bio-Rad, Hercules, CA). The high-intensity spots in the 3 corners are positive controls; the top left corners contain the negative controls. A graph of the relative intensity (compared with average of positive controls) is shown for selected cytokines. The inset graph is the same data, but on a larger scale for cytokines with higher expression levels. (B) PBTs were stimulated with 10 ng/mL OKT3 in the presence or absence of 10 nM dasatinib for 48 hours. Supernatants were collected for cytokine analysis as in Figure 4A, selected cytokines are labeled: red, cytokines that decreased by greater than 2-fold in the presence of dasatinib; green, cytokines that decreased by less than 2-fold or did not change in the presence of dasatinib.