Figure EV3. Licochalcone B directly binds to NEK7 but does not affect the kinase activity of NEK7.

• A
  Cell lysates of PMA‐primed THP‐1 treated with nigericin or not were incubated with sepharose or LicoB‐sepharose. The pull‐down samples and input were analysed by Western blot.
• B
  Cell lysates of PMA‐primed THP‐1 were incubated with sepharose or LicoB‐sepharose in the presence of different concentrations of free LicoB (0.5 and 1 mM). The pull‐down samples and input were analysed by Western blot.
• C
  Cell lysates of LPS‐primed hPBMCs treated with nigericin or not were incubated with sepharose or LicoB‐sepharose. The pull‐down samples and input were analysed by Western blot.
• D
  Cell lysates of LPS‐primed hPBMCs were incubated with sepharose or LicoB‐sepharose in the presence of different concentrations of free LicoB (0.5 and 1 mM). The pull‐down samples and input were analysed by Western blot.
• E
  NEK7 was incubated with β‐casein and ATP in the presence of different concentrations of LicoB. NEK7 kinase activity was measured using an ADP‐based phosphatase coupled kinase assay.
• F
  Cell lysates of LPS‐primed BMDMs treated with nigericin or not were incubated with Sepharose, Sepharose‐LicoA or Sepharose‐LicoB. The pull‐down samples and input were analysed by Western blot.
• G, H
  Human monocytes were treated with LicoB for 1 h, prior to stimulation with LPS (200 ng/ml) for 14 h. (G) Western blot analyses of pro‐caspase‐1 (p45), pro‐IL‐1β, NLRP3 and ASC in the whole cell lysate (WCL); cleaved IL‐1β (p17) in the culture SN of BMDMs were shown. Coomassie Blue staining was used as the SN loading control, while lamin B was used as the lysate loading control. IL‐1β secretion (H) in the supernatant were measured by ELISA.

Data information: Error bars, mean ± SEM from three biological replicates. **P < 0.01, ***P < 0.001 and n.s.: not significant (one‐way ANOVA with Dunnett's post hoc test).

Source data are available online for this figure.