Fig. 2.
Interaction of CD154 with CD40 on the cell surface of activated platelets leads to cleavage of CD154.

Interaction of CD154 with CD40 on the cell surface of activated platelets leads to cleavage of CD154.

(A) Platelets were either left unstimulated (○) or were activated with 0.2 U/mL thrombin for the indicated times, and the concentration of sCD154 in the supernatants was determined using a specific ELISA. The data are shown as mean values ± SD of duplicates and are representative of 4 independent experiments. The low background levels of sCD154 observed in unstimulated platelets probably reflect a minimal activation of platelets through the isolation and incubation procedures. ●, activated; ▪, activated plus mAb MOPC-21; ▴, activated plus mAb G28-5. (B) CD154 was immunoprecipitated from lysates and supernatants of unstimulated or activated (0.2 U/mL thrombin for 1 hour) platelets with mAb TRAP2 and detected by Western blotting with a CD154-specific antiserum. In parallel, platelet activation was performed in the presence of anti-CD40 mAb G28-5 (10 μg/mL). Unstim indicates unstimulated; Lys, lysate; Sup, supernatant. (C) Platelets were either left unstimulated or activated for the indicated time periods, and sCD154 was immunoprecipitated with a chimeric CD40-Ig reagent. The immunoprecipitate was subjected to SDS-PAGE and detected by Western blotting with a CD154-specific antiserum. Unstim indicates unstimulated.

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