Immunoprecipitation and Western Blot

After indicated nigericin stimulation for 1 h, mice peritoneal macrophages were lysed in an immunoprecipitation (IP) buffer mixed with PMSF and the cocktail. Then, these cell lysates were reacted to specific antibodies ASC or NEK7 and protein G plus-agarose overnight and washed four times with the IP buffer. Immunoprecipitates were eluted by boiling with 1% (w/v) SDS loading buffer.

The supernatants (SN) were immunoprecipitated with NLRP3 antibodies for 12 h at 4°C and protein A/G agarose for 2 h. The immunoprecipitants were washed six times with the IP buffer and boiled with 1% (w/v) SDS loading buffer for 10 min for immunoblot analysis.

For Western blot, stimulated macrophages were lysed with CLB (CST) supplemented with the cocktail and PMSF and subsequently centrifuged at 12,000 g at 4°C for 10 min. Protein concentrations were detected with a bicinchoninic acid assay (Pierce). An equal content of extracts was separated by SDS-PAGE and transferred onto 0.22-mm PVDF membranes (Merck Millipore).