Investigating NLRP3 Ubiquitination Interactome

Transfect Myc-NLRP3, HA-Ub, GFP-YAP, Flag-AMPK plasmids (all purchased from Shanghai Bioengineering) and proteasome inhibitor MG132 (0.5, 1.0, 2.0 μmol/L) (Y-13259, MCE, Shanghai, China) of different concentrations into HEK293T cells, add Protein A/G agarose beads (ab193262, Abcam) to remove non-specific proteins, then immunoprecipitate using anti-Myc antibody (#2272, 1/1000; CST, human and mouse). Next, wash the co-immunoprecipitated samples multiple times to remove non-specific proteins and impurities, retaining the ubiquitinated protein and its interacting protein complex. Then, use anti-Myc antibody (#2272, 1/1000; CST, human and mouse), anti-Flag antibody (MA1-91878, 1/1000; Sigma, USA), anti-HA antibody (#2367, 1/1000; CST, human and mouse), and anti-GFP antibody (#2956, 1/1000; CST, human and mouse) for immunoblotting detection [24 (link)].