Fig. 4.

Isoalantolactone suppresses the activation of the NF-κB pathway by downregulating TRAF6 ubiquitination. a HEK293 cells were transfected with TAB3, p65, or TRAF6 together with an NF-κB luciferase reporter plasmid. The cells were treated with or without IAL at different concentrations, and luciferase activity was measured. b–d The extent of TRAF6 ubiquitination in HEK293 cells transfected with Myc-TRAF6 alone, co-transfected with Myc-TRAF6, and b HA-Ub, c HA-UbK63, or (d) HA-UbK48 with or without 10 μM IAL treatment was determined by immunoprecipitation. Cell lysates were subjected to IP with an anti-Myc Ab, followed by western blot analysis with an anti-HA or anti-Myc Ab. Total cell lysates were used as the control (Input). e–g Statistical analysis of TRAF6 ubiquitination in (b–d) histograms. The data represent the mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01, and ***P < 0.001)