The assembly of the NLRP3 inflammasome complex required adaptor protein ASC and its oligomerization to bring the receptor and zymogen pro-caspase-1 into close proximity, which led to caspase-1 activation, and to the cleavage of pro-IL-1β and pro-IL-18 into their active cytokine and pyroptotic cell death (Fernandes-Alnemri et al., 2007). Therefore, in order to evaluate if ethanol, in comparison with other inflammasome stimuli, was capable of inducing ASC oligomerization, the cleavage of caspase-1 and the production of IL-1β and IL-18, astrocytes were treated with LPS, ATP (a positive control of NLRP3 activation) and ethanol (10, 50 mM). Figure 3 shows that ethanol and the LPS or ATP treatment promoted: (i) up-regulation of NLRP3; (ii) caspase-1 activation, as evidenced by the appearance of the 10 kDa active caspase-1-clevage peptide (Figure 3A); (iii) ASC oligomerization, as demonstrated by the notably presence of ASC dimers and some trimers (Fernandes-Alnemri and Alnemri, 2008; Figure 3B); (iv) the association between caspase-1 cleavage and NLRP3, as demonstrated by the Co-IP of both proteins with the appearance of the active p20/p10 caspase-1 in treated-astrocytes (Figure 3C); and (v)
