Fig. 7: S5 directly targeted USP14 at Ser404, Phe405, and Cys414 to inhibit its function. | Cell Death & Disease

Fig. 7: S5 directly targeted USP14 at Ser404, Phe405, and Cys414 to inhibit its function.

From: Typically inhibiting USP14 promotes autophagy in M1-like macrophages and alleviates CLP-induced sepsis

Fig. 7

a Molecular docking analysis of S5 and USP14 at different domains. b The shape and polarity of S5 binding pocket surface. c, d RAW264.7 cells were incubated with DMSO or S5 for 6 h, then the thermal stabilization of USP14 were analyzed by CETSA at different temperatures (c) and series of concentrations (d). e To further determine the interaction between S5 and USP14, MST experiment was carried out with a series concentration of S5 and EGFP-USP14 or EGFP-USP14 mutant (S404A, F405A, and C414A). Results showed that the Kd value of binding affinity for S5 and EGFP-USP14 was 11.5 μM. And the binding capacity between S5 and USP14 mutant (S404A, F405A, and C414A) was obviously blocked. f Ub-AMC hydrolysis assay was performed to evaluate effect of S5 on deubiquitinating enzymic activity of USP14 according to the instruction described in “Materials and methods” section. Fluorescence intensity was recorded every 30 s for 20 min. Each experiment was repeated three times, and the average value was calculated.

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