Fig. 5: TRIM8 promotes the K48-linked ubiquitination and proteasomal degradation of HNF1α.

The expressions of HNF1α protein were detected in the Huh7 cells transfected with the plasmid overexpressing TRIM8 (Flag-TRIM8) and the control vector (A) or the Huh7 cells transfected with siTRIM8 and siNC (B). C Huh7 cells were infected with control or Flag-TRIM8 plasmids and then treated with cycloheximide (CHX; 20 μg/mL) for 0 h, 2 h, 4 h, and 6 h. WB analysis was performed to evaluate HNF1α protein levels. D Semi-quantification analysis of HNF1α protein levels after TRIM8 overexpression based on CHX-treated assay. E WB analysis of HNF1α in Huh7 cells after treatment with MG132 (20 µM) for 0 h, 1 h, 2 h, and 3 h. F Huh7 cells were infected with siNC or siTRIM8 and then treated with CHX (20 μg/mL) for 0 h, 2 h, 4 h, and 6 h, and WB was performed to evaluate HNF1α protein levels. G Semi-quantification analysis of HNF1α protein levels after TRIM8-knockdown based on CHX-treated assay. H MG132 (20 µM) was applied to Huh7 cells transfected with V5-HNF1α, HA-Ub, TRIM8 plasmids or control plasmids, and 8 h later, ubiquitination assays were performed to determine the ubiquitination levels of HNF1α. I Ubiquitination assays determined the ubiquitination of endogenous HNF1α in Huh7 cells transfected with plasmid overexpressing TRIM8 or the control plasmid. J The ubiquitination levels of HNF1α after TRIM8 overexpression was examined in Huh7 cells co-transfected with wild-type (WT) and mutated ubiquitin (K48O, K48R, K63R) plasmids. Experiments were performed in triplicate and data are presented as means ± SEM. ns no significance, *P < 0.05 and **P < 0.01 using two- tailed Student’s t tests.