Rabbit anti human caspase 1 sc 515

For immunoblotting, cells were lysed with buffer (10 mM Tris pH 7.5, 1% NP-40, 150 mM NaCl, and protease inhibitor cocktail). Proteins were separated on sodium dodecyl sulphate-polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% milk in 1 X TBS with 0.5% Tween-20 and then probed with primary antibodies as follows: rabbit anti-human mature (17 kDa) IL-1β (D116, Cell Signaling, USA), goat anti-human pro-IL-1β (31 kDa) (sc-1250, Santa Cruz, USA), rabbit anti-human caspase-1 (sc-515, Santa Cruz, USA), and monoclonal mouse anti-human β-actin (KM9001, Tianjin Sungene Biotech, China). Appropriate HRP-conjugated secondary antibodies were used and signals were detected using ECL reagent (Amersham, USA).

THP-1 macrophages were infected with EV71 in RPMI 1640 medium without FBS. Cell lysates and chloroform-methanol concentrated supernatants were separated by SDS-PAGE, transferred to nitrocellulose membranes and hybridized with various primary antibodies. Rabbit anti-EV71 VP1 polyclonal antibody was generated previously^{37 (link)}. Rabbit anti- ASC (sc-22514, Santa Cruz), rabbit anti-human caspase-1 (sc-515, Santa Cruz), rabbit anti- mature and pro-IL-1β (sc-7884, Santa Cruz), mouse anti-β-actin (Sigma), as well as appropriate HRP-conjugated secondary antibodies (Santa Cruz) were applied for signal detection via ECL reagent (Perkin Elmer). ASC oligomerization detection was performed as described before^{25 (link)}.