Effects of ablation of HERC3 and/or RNF5/185 on the ERAD of ∆F508-NBD1, N1303K-CFTR, and ∆Y490-ABCB1-∆M1, correlation of the ERAD between ∆F508-CFTR and CFTR fragments. (A and B) The HiBiT degradation assay measured the ERAD of ∆F508-NBD1-HiBiT (A, n = 4) and N1303K-CFTR-HiBiT (B, n = 3) in 293MSR WT and RNF5/185 KO cells transfected with 50 nM siNT or siHERC3 as indicated. Two-way RM ANOVA revealed a significant main effect of HERC3 KD (A and B) or RNF5/185 DKO (B), but no interaction between them (P_int > 0.05, in A and B). (C–E) The correlation of ERAD rates between ∆F508-CFTR (Fig. 3 F) and M1 (C, Fig. 9 B), M1-N1(∆F) (D, Fig. 9 C), or M2 (E, Fig. 9 D) in 293MSR WT and RNF5/185 KO cells transfected with 50 nM siNT or siHERC3. (F) The HiBiT degradation assay measured the ERAD of ∆Y490-ABCB1-∆M1-HiBiT in 293MSR WT and RNF5/185 KO cells transfected with 50 nM siNT or siHERC3 as indicated (n = 3). Two-way RM ANOVA revealed a significant main effect of RNF5/185 DKO, but not that of HERC3 KD, and no interaction between them. Each biological replicate (n) is color-coded: the averages from four technical replicates are shown in triangles (A, B, and F). Data represent mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001, ns, not significant.