NLRP3 oligomerization

BMDMs were primed with LPS (100 ng/ml) for 4 h followed by 1-h nigericin(10 μM) stimulation. Cells were washed with cold PBS and resuspended in an ice-cold buffer (20 mM HEPES-KOH, pH 7.5, 150 mM KCl, 5 mM EDTA, and protease inhibitor), and lysed by passing 20 times through a 21-G needle. Lysates were centrifuged at 900 g for 8 min to remove nuclei and unlysed cells. Supernatants were then centrifuged at 6,200 g for 8 min. The pellet fractions were washed twice with PBS and then cross-linked with 2 mM fresh Suberic acid bis (3-sulfo-N-hydroxysuccinimide ester) sodium salt (BS3, Sigma,82436-77-9) for 1 h at 37°C and dissolved in SDS loading buffer to stop the cross-linking. The cross-linked pellets were separated in SDS–PAGE, and immunoblotting was performed.