Fig. 4

CB1R assembled multiple pyroptosis proteins via amino acids 177–209 and prevented the NLRP3 inflammasome from degradation. a, b In the Cb1r knockout HL-1 cells or the Cb1r stably overexpressed HL-1 cells, Olanzapine (Olz, 4 μM) with or without the specific protein synthesis inhibitor (cyclohexane, CHX, 200 nM) were added to treat cells for indicated hours. Western blot was performed to detect the protein levels of pyroptotic proteins. c In the Cb1r knockout HL-1 cells or the Cb1r stably overexpressed HL-1 cells, co-immunoprecipitation (Co-IP) assay was performed to detect the endogenous physical interaction between CB1R with pyroptotic proteins. d A flag-tagged Cb1r plasmid was co-transfected with each of the Myc-tagged Nlrp3 plasmid, His-tagged Gsdmd plasmid, HA-tagged Casp1 plasmid, and the GST-tagged Asc plasmid in HL-1 cells. Co-IP assay was performed to detect exogenous interaction of CB1R with the NLRP3 inflammasome. e Schematic illustration of mouse CB1R with full length (CB1R-FL, 1-473 amino acids), and truncated domains including Δ1-23 (CB1R-Mutant 1, or CB1R-M1), Δ1-142 (CB1R-M2), Δ1-176 (CB1R-M3), Δ1-209 (CB1R-M4), Δ1-209 (CB1R-M5), Δ1-254 (CB1R-M5), Δ1-299 (CB1R-M6), Δ1-366 (CB1R-M7), Δ1-399 (CB1R-M8). f The CB1R-FL, eight truncation mutants and three point-mutants were individually transfected into HL-1 cells. Western blot assay was performed to detect the protein levels of pyroptotic proteins. g The CB1R-M3 and CB1R-M4 plasmids were individually transfected into HEK293T cells in combination with each of the recombinant pyroptosis plasmids. Co-IP assay was conducted to detect protein interaction. h The simulated interaction diagram of human CB1R peptide (amino acids 177-189) with human proteins NLRP3 (up, pink cartoon), CASP1 (middle, yellow cartoon), or GSDMD (down, gray cartoon) in left panels. Right panels showed the CB1R peptide (blue) and key NLPR3 (green), CASP1 (orange), and GSDMD (purple) residues involved in ligand binding in stick representation