Il 6 elisas

After macrophages were primed with Pam3 and challenged with 5 μg/ml PGPC, POVPC, or LPC, >80% of cells were alive. Culture medium was collected, medium protein was precipitated using the chloroform-methanol method described by Fic et al., 2010 (link). The protein pellet was dissolved in SDS loading buffer containing 10% β-mercaptoethanol and then subjected for western blot of caspase 1 (p20). The cell lysate was used for actin detection. When macrophages were treated with 12.5 μg/ml PGPC or POVPC, ~80% cells died after 18 hr treatment; the culture medium and cells were combined, concentrated, and subjected to western blot analysis of IL-1β (p17), caspase 1 (p20), and actin. All of the concentrated proteins from each well were used for western blot. IL-1β (p17), caspase 1 (p20), and actin were detected using monoclonal antibodies D4T2D, E2G2I (Cell Signaling Technology), and 2D4H5 (Proteintech), respectively. The western blots were quantitated using ImageJ (NIH). Mouse IL-1β (R&D Systems), TNF-α, and IL-6 ELISAs (BD) were performed according to the manufacturer’s instructions.