Figure 6. Comparison of MHC-II membranes released from immature and mature BMDC via constitutive or ATP-stimulated mechanisms.

A. BMDC derived from C57BL/6 mice were primed with LPS (1 μg/ml) for 4 h, transferred to BSS, and stimulated with or without 5 mM ATP for 15 min. The extracellular media and the cell lysates were processed and analyzed by western blot sequentially probed with antibodies against MHC-II, caspase-1, IL-1β, and LAMP-1. The results are representative of observations from 3 experiments. B. C. D. BMDC were primed either without (for immature DC) or with 1 μg/ml LPS (for mature DC) for 24 h before further experimentation. B. Immature or mature DC was incubated in serum-free DMEM media for 24 hr before collection and processing of the extracellular fraction. C. D. Mature DC were transferred to BSS and stimulated either with or without 5 mM ATP for 20 min before collection and processing of the extracellular fraction. The collected extracellular media were sequentially centrifuged to eliminate dead cells and debris, followed by concentration. In panels B and C, the concentrated samples were directly loaded onto linear sucrose gradients. D. The concentrated medium samples were supplemented with or without Triton X-100 before loading onto linear sucrose gradients. The sucrose gradient samples were then centrifuged at 100,000×g for 16 h. 1 ml fractions were collected, weighed, precipitated by TCA, and analyzed by western blot.