Fig. 2

FKB interacts with the NAE1 to inhibit Ubc12 neddylaton. a, Autodocktools program was used to dock FKB with the NAE1 protein (3gzn), which has a predicted IC₅₀ of 986.1 nM, and Pymol program was used to observe the superimposed binding of FKB to NAE1. FKB was predicted to bind within the E1 regulatory subunit (grey) of NAE1. b, In vitro Ubc12 neddylation initiation assay was performed. Ubc12 antibody was used to detect the changes of NEDD8- conjugated Ubc12 bands, which are inhibited by FKB treatments. Densitometry analysis of Western blotting bands shows an estimated IC₅₀ of ~ 11 μM for FKB to inhibit Ubc12 neddylation. c, Cellular thermal shift assay was performed using PC3 cells treated with 8.8 μM FKB or 0.1% DMSO under a range of temperatures from 40^cC to 62 °C. Western blotting bands were semi-quantified by densitometry analysis and adjusted by loading control β-tubulin. The line graph shows relative changes of density ratios from 40^cC to 62 °C. Error bars represent standard deviations of three replicates. d. Cellular isothermal dose response was examined on PC3 cells at 58 °C and treated with FKB at concentrations ranged from 0.3125 μM to 20 μM, where 0.1% DMSO was used as a vehicle control. The line graph shows relative changes of density ratios from different concentrations of FKB treatment relative to vehicle control. CT denotes control. Error bars represent standard deviations of three replicates