FIGURE 5.
cAMP-mediated TNFR1 exosome-like vesicle release requires BIG2. HUVEC were transfected with vehicle (Mock); DMSO (Vehicle), 100 nm control, nontargeting siRNA (Control); or 100 nm siRNA targeting BIG2, without or with 1 mm cAMP for 3 days prior to the addition of fresh, exosome-depleted medium for 24 h. A, TNFR1 concentration in medium was quantified by ELISA. *, significant reduction in the quantity of TNFR1 present in medium from cells treated with cAMP and siRNA targeting BIG2 as compared with those treated with cAMP alone (p < 10-13, n = 6). B, representative Western blot, from one of three experiments, showing TNFR1 in medium and cell lysates and showing BIG2 and β-tubulin in cell lysates. C, densitometry was performed on the Western blots from B, and the quantity of the 55-kDa TNFR1 that was released into conditioned medium is presented as arbitrary densitometry units. *, significant decrease in the quantity of the 55-kDa TNFR1 present in conditioned medium from cells transfected with siRNA targeting BIG2 as compared with those treated with cAMP alone (p = 0.006, n = 3).