Fig 1: Bigelovin targets the RACK1 protein. A) Workflow of isoDTB‐ABPP experiment. B) Bigelovin binding proteins identified by isoDTB‐ABPP experiment. C–E) LPS‐primed BMDMs were incubated with DMSO or bigelovin (300 nM) for 1 h, and cellular thermal shift assays (CETSA) analyzed the thermal stabilization of Agfg2 (C), Sash1 (C), Gbp4 (D), RACK1 (E) at different temperatures (n = 3). F) LPS‐primed BMDMs were incubated with different concentration of bigelovin for 1 h, then thermal stability of RACK1 protein was evaluated by CESTA at 49 °C (n = 3). G) Mass spectrometry analysis of the binding site of bigelovin to RACK1. H) HEK‐293T cells transfected with indicated plasmid were incubated with bigelovin (300 nM) for 24 h, then thermal stability of GFP‐RACK1 and GFP‐RACK1 (C168A) protein were evaluated by CESTA at different temperature (n = 3). I) Mice were administrated with DMSO or bigelovin (0.1 mg kg−1) for 3 days by intraperitoneal injection and then challenged with 1% starch broth solution for 48 h, thermal stability of RACK1 protein in peritoneal macrophages was evaluated by CESTA at different temperature (n = 3). J) The interaction of bigelovin with RACK1 was measured by SPR. K) The interaction between bigelovin and RACK1 was measured by molecular docking.
Fig 2: Bigelovin inhibits NLRP3 inflammasome by binding to cysteine 168 of RACK1. A,B) LPS‐primed BMDM was treated with bigelovin and then stimulated with ATP (5 mM, 30 min). Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies (A). Quantitative analysis of the interaction of NLRP3‐RACK1 described in (A) (n = 3) (B). C,D) HEK‐293T cells were transfected FLAG‐NLRP3 with GFP‐Vector or GFP‐RACK1 and treated with bigelovin (300 nM) for 24 h. Cell lysates were immunoprecipitated and immunoblotted for analysis of the interaction of FLAG‐NLRP3 and GFP‐RACK1 (C). Quantitative analysis of the interaction of GFP‐RACK1‐FLAG‐NLRP3 described in (C) (n = 3) (D). E,F) HEK‐293T cells were transiently transfected with and either GFP‐RACK1 or GFP‐RACK1 (C168A) and treated with bigelovin (300 nM) for 24 h. Samples were immunoprecipitated with FLAG and probed for FLAG (NLRP3) and GFP (RACK1 or RACK1 (C168A) (E). Quantitative analysis of the immunoblot of GFP‐RACK1/FLAG‐NLRP3 described in 9(E) (n = 3) (F). G,H) Immunoprecipitation analysis of NLRP3‐NLRP3 interaction in HEK‐293T cells transfected with FLAG‐NLRP3, GFP‐NLRP3, GFP‐RACK1 or GFP‐RACK1 mutant (C168A) plasmid and treated with bigelovin (300 nM) for 24 h (G). Quantitative analysis of the interaction GFP‐NLRP3‐FLAG‐NLRP3 described in (G) (n = 3) (H). I,J) Mice were intravenously injected with siRNA (80 µg per mouse) with in vivo‐jetPEI, followed by intraperitoneal injection of 7.5 mg kg−1 LPS 48 h later, and then injected intraperitoneally bigelovin for 12 h. I) Western blotting analysis of RACK1 and caspase‐1 (p12) from above mice (n = 6). J) ELISA quantification of IL‐1β levels of lung tissues from above mice were measured (n = 6). Data were presented as mean ± SEM and statistical significance was assessed by two‐tailed unpaired t test.
from Biorbyt for Human RACK1 protein
Application Notes: Centrifuge the vial at 10,000 rpm for 1 minute, reconstitute at 200 µg/ml in sterile distilled water by gentle pipetting 2-3 times, don’t vortex