Figure 3.
HERC3 and RNF5/185 additively facilitate ∆F508-CFTR ERAD. (A) A schematic diagram of the HiBiT degradation assay, where ∆F508-CFTR-HiBiT(CT) and cytosolic LgBiT were co-expressed. The luminescence signal generated by the interaction of the HiBiT tag and LgBiT was measured in living cells. (B) A typical measurement of ∆F508-CFTR-HiBiT(CT) ERAD in 293MSR cells. The luminescence signal during the CHX chase was measured as the remaining ∆F508-CFTR during the CHX chase, with or without 10 µM MG-132. (C) The metabolic stability of ∆F508-CFTR-HiBiT(CT) was assessed through a CHX chase at 37°C, followed by Western blotting using an anti-HiBiT antibody in 293MSR cells (n = 2). The remaining ∆F508-CFTR was expressed as a percentage of time 0, and one-phase exponential decay curves were fitted. (D) Western blotting confirmed the ablation of RNF5, and RNF185 in the WT and RNF5/185 DKO 293MSR cells transfected with siRNA indicated. Ponceau staining was used as a loading control. (E) HERC3 KD in 293MSR WT and RNF5/185 DKO cells was confirmed through quantitative PCR (n = 3). (F) Kinetic degradation of ∆F508-CFTR-HiBiT(CT) in 293MSR WT and RNF5/185 KO cells transfected with 50 nM siNT or siHERC3. Luminescence was continuously monitored over 180 min in the presence of CHX and plotted normalized to the non-treated cells. The ERAD rate of ∆F508-CFTR-HiBiT(CT) was calculated by fitting the initial degradation portion of each kinetic degradation curve (right, n = 3). (G–I) Kinetic degradation of ∆F508-CFTR-Nluc(CT) in 293MSR (G, n = 4), BEAS-2B (H, n = 3), and CFBE (I, n = 12) cells transfected with 50 nM siRNA as indicated. The ERAD rate of ∆F508-CFTR-Nluc(CT) was calculated as F. Each biological replicate (n) is color-coded: the averages from three to four technical replicates are shown in triangles (E–H). Statistical significance was assessed by one-way RM ANOVA with Dunnett’s multiple comparison tests (E) or two-way RM ANOVA which revealed a significant main effect of HERC3 or RNF5/185 ablation, but no interaction between them (F–I, Pint> 0.05). 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 F3.

HERC3 and RNF5/185 additively facilitate ∆F508-CFTR ERAD. (A) A schematic diagram of the HiBiT degradation assay, where ∆F508-CFTR-HiBiT(CT) and cytosolic LgBiT were co-expressed. The luminescence signal generated by the interaction of the HiBiT tag and LgBiT was measured in living cells. (B) A typical measurement of ∆F508-CFTR-HiBiT(CT) ERAD in 293MSR cells. The luminescence signal during the CHX chase was measured as the remaining ∆F508-CFTR during the CHX chase, with or without 10 µM MG-132. (C) The metabolic stability of ∆F508-CFTR-HiBiT(CT) was assessed through a CHX chase at 37°C, followed by Western blotting using an anti-HiBiT antibody in 293MSR cells (n = 2). The remaining ∆F508-CFTR was expressed as a percentage of time 0, and one-phase exponential decay curves were fitted. (D) Western blotting confirmed the ablation of RNF5, and RNF185 in the WT and RNF5/185 DKO 293MSR cells transfected with siRNA indicated. Ponceau staining was used as a loading control. (E) HERC3 KD in 293MSR WT and RNF5/185 DKO cells was confirmed through quantitative PCR (n = 3). (F) Kinetic degradation of ∆F508-CFTR-HiBiT(CT) in 293MSR WT and RNF5/185 KO cells transfected with 50 nM siNT or siHERC3. Luminescence was continuously monitored over 180 min in the presence of CHX and plotted normalized to the non-treated cells. The ERAD rate of ∆F508-CFTR-HiBiT(CT) was calculated by fitting the initial degradation portion of each kinetic degradation curve (right, n = 3). (G–I) Kinetic degradation of ∆F508-CFTR-Nluc(CT) in 293MSR (G, n = 4), BEAS-2B (H, n = 3), and CFBE (I, n = 12) cells transfected with 50 nM siRNA as indicated. The ERAD rate of ∆F508-CFTR-Nluc(CT) was calculated as F. Each biological replicate (n) is color-coded: the averages from three to four technical replicates are shown in triangles (E–H). Statistical significance was assessed by one-way RM ANOVA with Dunnett’s multiple comparison tests (E) or two-way RM ANOVA which revealed a significant main effect of HERC3 or RNF5/185 ablation, but no interaction between them (F–I, Pint> 0.05). 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 F3.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal