HERC3 and RNF5/185 facilitate ∆F508-CFTR ubiquitination. (A) Ubiquitination levels of HBH-∆F508-CFTR-3HA in 293MSR WT and RNF5/185 DKO cells were measured by Neutravidin pull-down under denaturing conditions (NA pull-down) and Western blotting. The CFTR ubiquitination level was quantified by densitometry and normalized to CFTR in precipitates (right, n = 3). Two-way RM ANOVA revealed a significant main effect of RNF5/185 DKO and no interaction between HERC3 KD and RNF5/185 DKO (P_int > 0.05). (B and C) K48 (B, n = 3) and K63-linked polyubiquitination (C, n = 3) of HBH-∆F508-CFTR in 293MSR WT and RNF5/185 DKO cells transfected with 50 nM siNT or siHERC3 were quantified by Ub ELISA using Ub linkage-specific antibodies. 10 µM MG-132 was treated for 3 h at 37°C. The ubiquitination level was normalized by the CFTR amount quantitated by ELISA using an anti-HA antibody. Two-way RM ANOVA revealed significant main effects of HERC3 KD or RNF5/185 DKO and a significant interaction between them in H, but not in G (P_int > 0.05). (D) The effect of HERC3 KD on K48 and K63-linked poly-ubiquitination of HBH-∆F508-CFTR in RNF5/185 DKO cells was measured by Ub ELISA using higher amounts of cell lysate. Statistical significance was assessed by a two-tailed paired t test (n = 3). Each biological replicate (n) is color-coded: the averages from three or four technical replicates are shown in triangles (B–D). 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. Source data are available for this figure: SourceData F5.