The substrate selectivity of HERC3 in ERAD. (A) A schematic diagram of the ERAD substrate models used in this study. The misfolded region is indicated by a star. The HiBiT tag was fused in the cytoplasmic region except for D18G-TTR. (B and C) The HiBiT degradation assay measured the ERAD of TCRα-HiBiT (B, n = 4) and Insig-1-HiBiT (C, n = 3) in 293MSR WT and RNF5/185 KO cells transfected with 50 nM siNT or siHERC3, as indicated. (D) The metabolic stability of D18G-TTR was measured by CHX chase at 37°C and Western blotting with an anti-TTR antibody in 293MSR WT and RNF5/185 KO cells transfected with 50 nM siNT or siHERC3 as indicated. The remaining TTR was quantified by densitometry and expressed as a percentage of the initial amount (right, n = 3). (E) Western blotting analyzed the effects of Myc-HERC3 OE on co-transfected ∆F508-CFTR-3HA, TCRα-HA, D18G-TTR-FLAG, or WT-CFTR-3HA in COS-7 cells. The immature ∆F508-CFTR (B band), TCRα, D18G-TTR, and total WT-CFTR (B and C bands) were quantified by densitometry (n = 3). (F) The HiBiT degradation assay measured the ERAD of ∆Y490-ABCB1-HiBiT (E, n = 3) in 293MSR WT and RNF5/185 KO cells as B and C. Each biological replicate (n) is color-coded: the averages from four technical replicates are shown in triangles (B, C, and F). Statistical significance was assessed by a one-way RM ANOVA with Dunnett’s multiple comparison tests (E) or two-way RM ANOVA revealed no significant main effect of HERC3 KD or RNF5/185 DKO, and no interaction between them (P_int > 0.05), except for a significant main effect of RNF5/185 DKO in F. Data distribution was assumed to be normal but was not formally tested. Data represent mean ± SD. *P < 0.05, **P < 0.01, ns, not significant. Source data are available for this figure: SourceData F8.