Figure 7. UBQLN proteins facilitate... | Rockefeller University Press

Figure 7.

$UBQLN proteins facilitate ∆F508-CFTR retrotranslocation and ERAD. (A) Western blotting confirmed the triple KD of UBQLN1, 2, and 4 in 293MSR cells transfected with 50 nM siRNA as indicated. Ponceau staining was used as a loading control. (B) Kinetic degradation of ∆F508-CFTR-HiBiT(CT) in 293MSR WT cells transfected with 50 nM siNT or siUBQLN1/2/4. The ERAD rate was calculated by fitting the initial degradation portion of each kinetic degradation curve (right, n = 3). (C) Kinetic retrotranslocation of ∆F508-CFTR-HiBiT(Ex) in 293MSR cells upon UBQLN triple KD. The retrotranslocation was calculated by linear fitting (right, n = 3). (D) Kinetic ER disappearance of ∆F508-CFTR-HiBiT(Ex) in 293MSR cells upon UBQLN triple KD. The ER disappearance rate was calculated by fitting the kinetic ER disappearance curve (right, n = 3). (E) The detergent NP-40 solubility of ∆F508-CFTR-HiBiT(CT) in 293MSR cells was assessed following UBQLN1/2/4 triple KD or MG-132 treatment (10 µM, 3 h) using Western blotting with an anti-HiBiT antibody (n = 3). The soluble (100 µg) and insoluble (40 µg) fractions were analyzed. (F) The effects of overexpressed FLAG-UBQLN2 variants on the steady-state level of ∆F508-CFTR-3HA were analyzed by Western blotting in co-transfected COS-7 cells. The immature ∆F508-CFTR (B band) was quantified by densitometry (right, n = 3). A schematic diagram of the UBQLN2 domain composition with the residue numbers at the domain boundaries. UBQLN2 mutants used in this study are also shown. (G) The interaction of FLAG-UBQLN2 variants with HBH-∆F508-CFTR-3HA in BHK cells was analyzed by NA pull-down and Western blotting. Cells were treated with 10 µM MG-132 for 3 h before cell lysis. Statistical significance was assessed by a two-tailed paired t test (B–D), or one-way RM ANOVA with Dunnett’s multiple comparison tests (E and F). Each biological replicate (n) is color-coded: the averages from 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, ns, not significant. Source data are available for this figure: SourceData F7.$

UBQLN proteins facilitate ∆F508-CFTR retrotranslocation and ERAD. (A) Western blotting confirmed the triple KD of UBQLN1, 2, and 4 in 293MSR cells transfected with 50 nM siRNA as indicated. Ponceau staining was used as a loading control. (B) Kinetic degradation of ∆F508-CFTR-HiBiT(CT) in 293MSR WT cells transfected with 50 nM siNT or siUBQLN1/2/4. The ERAD rate was calculated by fitting the initial degradation portion of each kinetic degradation curve (right, n = 3). (C) Kinetic retrotranslocation of ∆F508-CFTR-HiBiT(Ex) in 293MSR cells upon UBQLN triple KD. The retrotranslocation was calculated by linear fitting (right, n = 3). (D) Kinetic ER disappearance of ∆F508-CFTR-HiBiT(Ex) in 293MSR cells upon UBQLN triple KD. The ER disappearance rate was calculated by fitting the kinetic ER disappearance curve (right, n = 3). (E) The detergent NP-40 solubility of ∆F508-CFTR-HiBiT(CT) in 293MSR cells was assessed following UBQLN1/2/4 triple KD or MG-132 treatment (10 µM, 3 h) using Western blotting with an anti-HiBiT antibody (n = 3). The soluble (100 µg) and insoluble (40 µg) fractions were analyzed. (F) The effects of overexpressed FLAG-UBQLN2 variants on the steady-state level of ∆F508-CFTR-3HA were analyzed by Western blotting in co-transfected COS-7 cells. The immature ∆F508-CFTR (B band) was quantified by densitometry (right, n = 3). A schematic diagram of the UBQLN2 domain composition with the residue numbers at the domain boundaries. UBQLN2 mutants used in this study are also shown. (G) The interaction of FLAG-UBQLN2 variants with HBH-∆F508-CFTR-3HA in BHK cells was analyzed by NA pull-down and Western blotting. Cells were treated with 10 µM MG-132 for 3 h before cell lysis. Statistical significance was assessed by a two-tailed paired t test (B–D), or one-way RM ANOVA with Dunnett’s multiple comparison tests (E and F). Each biological replicate (n) is color-coded: the averages from 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, ns, not significant. Source data are available for this figure: SourceData F7.

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