To detect NLRP3/ASC interaction, cells were lysed with IP buffer (1% NP-40, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, and complete protease and phosphatase inhibitor cocktail) and sonicated for 5 sec at 20 amp. The lysates were precleared and incubated with ASC antibody (sc-22514-R, Santa Cruz)46 (link) overnight at 4 °C46 (link).
To detect NLRP3 ubiquitination, BMDMs were lysed with TTNE. The lysates were precleared and incubated with NLRP3 antibody overnight at 4 °C. the beads were rinsed with lysis buffer and eluted (0.1 M Tris-HCl, pH 6.8, 0.4% sodium dodecyl sulfate, 12% glycerol, 0.286 M β-mercaptoethanol, and 0.32% bromophenol blue)35 (link). The lysates were precleared and incubated with NLRP3 antibody (Cryo-2, Adipogen)35 (link) overnight at 4 °C.
To detect NLRP3 ubiquitination, BMDMs were lysed with TTNE. The lysates were precleared and incubated with NLRP3 antibody overnight at 4 °C. the beads were rinsed with lysis buffer and eluted (0.1 M Tris-HCl, pH 6.8, 0.4% sodium dodecyl sulfate, 12% glycerol, 0.286 M β-mercaptoethanol, and 0.32% bromophenol blue)35 (link). The lysates were precleared and incubated with NLRP3 antibody (Cryo-2, Adipogen)35 (link) overnight at 4 °C.
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