Fig. 5

Hakai mediates Ajuba degradation via neddylation. a Immunoblot analysis and quantification of the half-life of Ajuba in the presence of cycloheximide (CHX, 80 μg/ml), and in the presence or absence of lactacystin (LAC, 20 μM) or MG132 (10 μM) in BEL7402 and HepG2 cells. GAPDH was used as a loading control. Data are presented as Mean ± SEM from three independent experiments (**p < 0.01, ***p < 0.001). b, c BEL7402 or HepG2 cells were transfected with GFP-tagged Hakai and treated with 0.5% DMSO, 20 μM LAC, 10 μM MG132 (b) or 5 μM MLN4924 (c). Endogenous Ajuba levels were determined by immunoblotting using anti-Ajuba antibody. GAPDH was used as a loading control. Densitometry was performed for quantification, and the ratios of Ajuba and GAPDH are presented. d Neddylation assay of Ajuba in 293 T cells transfected with the indicated plasmids. IB, immnoblot. IP, immunoprecipitation. WCL, Whole-cell lysates. e HepG2 cells were transfected with GFP-tagged Hakai or HYB domain deletion mutant and treated with 5 μM MLN4924 or 0.5% DMSO. Endogenous Ajuba protein levels were determined and quantified. GAPDH was used as a loading control. f Ectopic Ajuba protein levels were examined in 293 T cells transfected with Myc-tagged Ajuba, GFP-tagged Hakai or HYB domain deletion mutant in the presence or absence of 5 μM MLN4924. GAPDH was used as a loading control. g Colony formation assay performed in HepG2 cells infected with Adenoviruses expressing Hakai in presence or absence of 5 μM MLN4924. Data are presented as Mean ± SEM from three independent experiments (**p < 0.01, ***p < 0.001)