HERC3 selectively facilitates ERAD by interacting with the CFTR-MSDs. (A) A schematic diagram of the CFTR fragment models used in this study. The misfolded region is indicated by a star. M1; MSD1, M1-N1(∆F); MSD1 and NBD1 with ∆F508 mutation, M2; MSD2, N1(∆F); NBD1 with ∆F508 mutation. ∆Y490-ABCB1-MSD1^CFTR and ∆Y490-ABCB1-MSD2^CFTR are the chimeras in which the MSD1 and MSD2 of ABCB1 were replaced with respective MSDs of CFTR. The HiBiT tag was fused in the C-terminal region located in the cytoplasm. (B–D) The HiBiT degradation assay measured the ERAD of M1-HiBiT (B, n = 3), M1-N1(∆F)-HiBiT (C, n = 3), and M2-HiBiT (D, n = 3) in 293MSR WT and RNF5/185 KO cells transfected with 50 nM siNT or siHERC3, as indicated. (E and F) The HiBiT degradation assay measured the ERAD of ∆Y490-ABCB1, ∆Y490-ABCB1-∆M1, ∆Y490-ABCB1-∆M2, ∆Y490-ABCB1-M1^CFTR, and ∆Y490-ABCB1-M2^CFTR in 293MSR WT cells (F, n = 3). The ABCB1-HiBiT constructs analyzed were illustrated in E. (G and H) The HiBiT degradation assay measured the ERAD of ∆Y490-ABCB1-M1^CFTR (G, n = 4) and ∆Y490-ABCB1-M2^CFTR (H, n = 4) in 293MSR WT and RNF5/185 KO cells transfected with 50 nM siNT or siHERC3, as indicated. Statistical significance was assessed using a two-tailed paired t test (F) or two-way RM ANOVA (B–D, G, and H) which revealed a significant main effect of HERC3 KD or RNF5/185 DKO, but no significant interaction between them (P_int > 0.05), except for C. Each biological replicate (n) is color-coded, and the averages from four technical replicates are represented by triangles. Data distribution was assumed to be normal but was not formally tested. Data represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.