Journal: Cell reports. Medicine
Article Title: Entrectinib inhibits NLRP3 inflammasome and inflammatory diseases by directly targeting NEK7.
doi: 10.1016/j.xcrm.2023.101310
Figure Lengend Snippet: Figure 4. ENB binds to R121 of NEK7 (A) ELISA of IL-1b in SNs from LPS-primed BMDMs treated with various doses of ENB for 15 min and then washed three times and stimulated with 5 mM nigericin for 30 min. (B) Docking complex of NEK7 with ENB. ENB is shown as sticks and colored light green, NEK7 is showed in cartoon and colored light gray, and key amino acid residues are shown as sticks. (C) 2D binding mode diagrams of NEK7 and ENB. (D) DARTS and immunoblot analysis of NEK7 in HEK293T cells transfected with WT, I40A, or R121A mutant FLAG-NEK7 and then treated with 200 mM ENB and pronase (25 ng/mg of protein). (E) IP and immunoblot analysis of HEK293T cells transfected with VSV-NLRP3, WT, or R121A mutant FLAG-NEK7 plasmids as indicated and treated with 2 mM ENB. (F) ELISA analysis of IL-1b in SNs from NEK7 knockout THP-1 cells recombined with human WT or R121A mutant NEK7 and then stimulated with 5 mM nigericin for 1 h. (G) ELISA of IL-1b in SNs from NEK7 knockout iBMDMs recombined with mouse WT or R121A mutant NEK7 and then stimulated with 5 mM nigericin for 2 h. (H) Immunoblot analysis of P20 in SNs, pro-casp1, and NEK7 in input from NEK7 knockout iBMDMs recombined with mouse WT or R121A mutant NEK7 and then stimulated with 5 mM nigericin for 2 h. Data are representative of one replicate of five independent experiments and show as mean ± SEM. Statistical analysis was performed using two-way ANOVA (A, F, and G). *p < 0.05, ***p < 0.001; NS, not significant, p > 0.05.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse IL-1b R&D Systems Cat#AF-401-NA; RRID:AB_416684 Anti-mouse caspase-1 Adipogen Cat#AG-20B-0042; RRID:AB_2490248 Anti-human IL-1b Proteintech Cat#60136-1-Ig; RRID:AB_10597543 Anti-human caspase-1 Cell Signaling Technology Cat#2225; RRID:AB_2243894 Anti-NLRP3 Adipogen Cat#AG-20B-0014; RRID:AB_2885199 Anti-b-actin Abmart Cat#P30002; RRID:AB_2222847 Anti-ASC Cell Signaling Technology Cat#67824; RRID:AB_2799736 Anti-NEK7 Santa Cruz Cat#SC-50756; RRID:AB_2235871 Anti-NEK6 Abcam Cat#ab133494; RRID:AB_11155676 Anti-NEK9 Abcam Cat# ab18332, RRID:AB_444424 Anti-GSDMD Abcam Cat#ab219800; RRID:AB_2888940 Anti-VSV Sigma Cat#V4888; RRID:AB_261872 Anti-Flag Sigma Cat#F2555; RRID:AB_796202 Chemicals, peptides, and recombinant proteins Entrectinib TargetMol Cat#T3678 Repotrectinib TargetMol Cat#T4071 Belizatinib TargetMol Cat#T4257 Larotrectinib TargetMol Cat#T5995 Val-boroPro TargetMol Cat#T37861 MSU Sigma Cat#69-93-2 ATP Sigma Cat#34369-07-8 PMA (phorbol-12-myristate-13-acetate) Sigma Cat#P8139 Poly (dA:dT) Sigma Cat#86828-69-5 Protein G agarose Sigma Cat#P7700 Propidium iodide (PI) Sigma Cat#P4170 b-hydroxybutyrate (BHB) Sigma Cat#52017 Pronase Sigma Cat#P5147 Imiquimod MedChemExpress Cat#HY-B0180 Nigericin MedChemExpress Cat#HY-127019 MCC950 MedChemExpress Cat#HY-12815 CY-09 MedChemExpress Cat#HY-103666 Tranilast MedChemExpress Cat#HY-B0195 Tivantinib MedChemExpress Cat#HY-50686 b-Estradiol MedChemExpress Cat#HY-B0141 Alum Thermo Fisher Scientific Cat#77161 Mitotracker Thermo Fisher Scientific Cat#M7512 Pam3CSK4 Invivogen Cat#tlrl-pms LPS Invivogen Cat#tlrl-peklps MitoSOX Invitrogen Cat#M36008 DAPI Invitrogen Cat#D21490 MQAE Invitrogen Cat#162558-52-3 Lipofectamine 2000 Invitrogen Cat#11668019 SiO2 InvivoGen Cat#tlrl-sio-2 (Continued on next page) e1 Cell Reports Medicine 4, 101310, December 19, 2023
Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Western Blot, Transfection, Mutagenesis, Knock-Out
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R&D Systems manufactures this product 
![Figure 2. ENB specifically inhibits NLRP3 without affecting activation of other inflammasomes (A) Immunoblot analysis of IL-1b and P20 in SNs and pro-IL-1b and pro-casp1 in input from LPS-primed BMDMs treated with 2 mM ENB and then stimulated with 5 mM nigericin for 30 min or transfected with 0.5 mg/mL poly(dA:dT) for 4 h. (B) ELISA of IL-1b in SNs from LPS-primed BMDMs treated with 2 mM ENB and then stimulated with 5 mM nigericin for 30 min or transfected with 0.5 mg/mL poly(dA:dT) for 4 h. (C) Immunoblot analysis of IL-1b and P20 in SNs and pro-IL-1b and Pro-casp1 in input from LPS-primed BMDMs treated with 2 mM ENB and then stimulated with 5 mM nigericin for 30 min or infected with Salmonella (multiplicity of infection [MOI] = 5) for 4 h. (D) ELISA of IL-1b in SNs from LPS-primed BMDMs treated with 2 mM ENB and then stimulated with 5 mM nigericin for 30 min or infected with Salmonella (MOI = 5) for 4 h. (E) ELISA of IL-1b in SNs from LPS-primed BMDMs treated with 2 mM ENB and then stimulated with 5 mM nigericin for 30 min or 0.5 mg/mL C. difficile toxin B (TcdB) for 1 h or infected with L. monocytogenes (Listeria; MOI = 10) for 4 h. (F) ELISA of IL-1b in SNs from phorbol 12-myristate 13-acetate (PMA)-differentiated and LPS-primed THP-1 cells treated with 2 mM ENB and then stimulated with 5 mM nigericin for 1 h or 10 mM Val-boroPro (VbP) for 24 h. (G) Immunoblot analysis of NLRP3 and pro-IL-1b in input from BMDMs treated with various doses of ENB and then stimulated with 50 ng/mL LPS for 3 h. (H and I) ELISA of TNF-a (H) and IL-6 (I) in SNs from BMDMs treated with various doses of ENB and then stimulated with 50 ng/mL LPS for 3 h. Data represent the mean ± SEM of four technical replicates from one of five independent experiments. Statistical analysis was performed using two-way ANOVA (B and D–F). ***p < 0.001.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_8409/pm38118409/pm38118409__page5_image1.jpg)
