Fig. 6: TRIM8 promotes the ubiquitination of HNF1α at Lys197.

A Schematic illustration of tandem ubiquitin-binding entity (TUBE) pull-down assay. B Huh7 cells transfected with V5-HNF1α or V5-HNF1α-K197R plasmids were treated with CHX (20 µg/ml) for 0, 2, 4, and 6 h, and the expression of HNF1α was analyzed by Western blotting (WB) assays. C Semi-quantification analysis of HNF1α protein levels in the indicated groups. D Ubiquitination assays were performed to determine the ubiquitination levels of HNF1α in Huh7 cells transfected with V5-HNF1α or V5-HNF1α-K197R and HA-Ub plasmids together with Flag-TRIM8 plasmids or negative control plasmids. E WB analysis of HNF1α protein levels in Huh7 cells transfected with HNF1α or HNF1α-K197R together with TRIM8 or its control plasmids. F Luciferase reporter assays were performed to evaluate the activity of the HNF1α promoter in Huh7 cells transfected with the indicated plasmids. G Relative mRNA levels of HNF1α target genes in MHCC-L cells infected with control lentivirus or lenti-TRIM8 together with lentivirus expressing HNF1α or HNF1α-K197R. Experiments were performed in triplicate and data are presented as means ± SEM. ns no significance, *P < 0.05, **P < 0.01, ***P < 0.001 using two- tailed Student’s t tests.