Figure 6. HERC3 facilitates ∆F508-CFTR... | Rockefeller University Press

Figure 6.

$HERC3 facilitates ∆F508-CFTR interaction with UBQLN2. (A) Western blotting showed the steady-state level of ΔF508-CFTR-3HA under OE of FLAG-UBQLN1 or FLAG-UBQLN2 in transiently coexpressed COS-7 cells. The CFTR level was quantified by densitometry (right, n = 4). Na+/K+ ATPase (ATPase) was used as a loading control. B, immature form. (B) The interaction between FLAG-UBQLN2 and HBH-∆F508-CFTR-3HA in BHK cells transfected with or without Myc-HERC3 was assessed using NA pull-down and Western blotting. The amount of UBQLN2 bound to HBH-∆F508-CFTR-3HA was quantified by densitometry and normalized to CFTR levels in the precipitates (right, n = 3). (C) The effect of Myc-HERC3 OE on the FLAG-UBQLN2 and HBH-∆F508-CFTR-3HA interaction in 293MSR WT cells was measured by ELISA using an anti-FLAG antibody. The level of FLAG-UBQLN2 binding was normalized to the CFTR level, which was measured by ELISA using an anti-HA antibody (n = 5). (D and E) The interaction between FLAG-UBQLN2 and HBH-∆F508-CFTR-3HA in 293MSR WT and RNF5/185 DKO cells transfected with 50 nM siNT or siHERC3 was measured by ELISA as C (D, n = 4). Additionally, under conditions of increased FLAG-UBQLN2 expression, the UBQLN2 binding to HBH-∆F508-CFTR-3HA in RNF5/185 DKO cells was quantified by ELISA (E, n = 3). (F) The association of HBH-∆F508-CFTR with endogenous UBQLN2 in 293MSR WT or RNF5/185 DKO cells transfected with 50 nM siNT or siHERC3 was analyzed by NA pull-down after DSP cross-linking. The quantities of UBQLN2 and ∆F508-CFTR in the precipitates were measured using densitometry and expressed as a percentage of the control. The quantities of CFTR-bound UBQLN2 were normalized to CFTR levels as UBQLN2/CFTR and expressed as a percentage of the control. (G) The level of endogenous UBQLN2 in the microsomes of 293MSR WT and RNF5/185 DKO cells transfected with 50 nM siNT or siHERC3 was measured. Cells were treated with or without 10 µM MG-132 for 3 h before subcellular fractionation. Microsomes enriched with ER membranes were confirmed using an anti-calnexin (CNX) antibody. The quantities of the ER-recruited UBQLN2 were quantified by subtracting the amount of UBQLN2 before MG-132 treatment from the amount after MG-132 treatment and were expressed as a percentage of the control (n = 4, right). Each biological replicate (n) is color-coded: the averages from three technical replicates are shown in triangles (C–E). Statistical significance was assessed by one-way RM ANOVA with Dunnett’s multiple comparison tests (A and C), a two-tailed paired t test (B and E), or two-way RM ANOVA (D and G). Data distribution was assumed to be normal but was not formally tested. Data represent mean ± SD. *P < 0.05, ***P < 0.001, ****P < 0.0001, ns, not significant. Source data are available for this figure: SourceData F6.$

HERC3 facilitates ∆F508-CFTR interaction with UBQLN2. (A) Western blotting showed the steady-state level of ΔF508-CFTR-3HA under OE of FLAG-UBQLN1 or FLAG-UBQLN2 in transiently coexpressed COS-7 cells. The CFTR level was quantified by densitometry (right, n = 4). Na⁺/K⁺ ATPase (ATPase) was used as a loading control. B, immature form. (B) The interaction between FLAG-UBQLN2 and HBH-∆F508-CFTR-3HA in BHK cells transfected with or without Myc-HERC3 was assessed using NA pull-down and Western blotting. The amount of UBQLN2 bound to HBH-∆F508-CFTR-3HA was quantified by densitometry and normalized to CFTR levels in the precipitates (right, n = 3). (C) The effect of Myc-HERC3 OE on the FLAG-UBQLN2 and HBH-∆F508-CFTR-3HA interaction in 293MSR WT cells was measured by ELISA using an anti-FLAG antibody. The level of FLAG-UBQLN2 binding was normalized to the CFTR level, which was measured by ELISA using an anti-HA antibody (n = 5). (D and E) The interaction between FLAG-UBQLN2 and HBH-∆F508-CFTR-3HA in 293MSR WT and RNF5/185 DKO cells transfected with 50 nM siNT or siHERC3 was measured by ELISA as C (D, n = 4). Additionally, under conditions of increased FLAG-UBQLN2 expression, the UBQLN2 binding to HBH-∆F508-CFTR-3HA in RNF5/185 DKO cells was quantified by ELISA (E, n = 3). (F) The association of HBH-∆F508-CFTR with endogenous UBQLN2 in 293MSR WT or RNF5/185 DKO cells transfected with 50 nM siNT or siHERC3 was analyzed by NA pull-down after DSP cross-linking. The quantities of UBQLN2 and ∆F508-CFTR in the precipitates were measured using densitometry and expressed as a percentage of the control. The quantities of CFTR-bound UBQLN2 were normalized to CFTR levels as UBQLN2/CFTR and expressed as a percentage of the control. (G) The level of endogenous UBQLN2 in the microsomes of 293MSR WT and RNF5/185 DKO cells transfected with 50 nM siNT or siHERC3 was measured. Cells were treated with or without 10 µM MG-132 for 3 h before subcellular fractionation. Microsomes enriched with ER membranes were confirmed using an anti-calnexin (CNX) antibody. The quantities of the ER-recruited UBQLN2 were quantified by subtracting the amount of UBQLN2 before MG-132 treatment from the amount after MG-132 treatment and were expressed as a percentage of the control (n = 4, right). Each biological replicate (n) is color-coded: the averages from three technical replicates are shown in triangles (C–E). Statistical significance was assessed by one-way RM ANOVA with Dunnett’s multiple comparison tests (A and C), a two-tailed paired t test (B and E), or two-way RM ANOVA (D and G). Data distribution was assumed to be normal but was not formally tested. Data represent mean ± SD. *P < 0.05, ***P < 0.001, ****P < 0.0001, ns, not significant. Source data are available for this figure: SourceData F6.

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