Figure 1.
HERC3 participates in the ubiquitination and ERAD of ∆F508-CFTR. (A) The PM density of r∆F508-CFTR-HRP in CFBE Teton cells transfected with 50 nM siNT or siHERC3, as indicated (n = 9–12). Each independent experiment, consisting of three to four biological replicates (n), is color-coded. (B) Quantitative PCR analysis assessed HERC3 KD efficiency in CFBE Teton ∆F508-CFTR-HRP cells (n = 3). Each biological replicate (n) is color-coded: the averages from three technical replicates are shown in triangles. (C) The channel function of r∆F508-CFTR-3HA in CFBE Teton cells transfected with 50 nM siNT or siHERC3 pool was measured by YFP quenching assay. The initial YFP quenching rate was quantified as the CFTR function (right, n = 19). Each independent experiment, consisting of four to eight biological replicates (n), is color-coded. (D) Western blotting analyzed steady-state levels of ∆F508-CFTR-3HA with (r∆F508) or without 26°C rescue (∆F508) in CFBE Teton cells transfected with 50 nM siNT or siHERC3 pool. Na+/K+ ATPase (ATPase) was used as a loading control. B, immature form; C, mature form. Western blotting also confirmed HERC3 KD in CFBE Teton ΔF508-CFTR-3HA cells. Ponceau staining was used as a loading control. A filled triangle indicates HERC3. (E) Cellular ∆F508-CFTR-3HA stability in CFBE Teton cells transfected with 50 nM siNT or siHERC3 pool was measured by cell-based ELISA using an anti-HA antibody after CHX treatment (n = 12). (F) The PM stability of r∆F508-CFTR-3HA in CFBE cells transfected with 50 nM siNT, siRFFL pool, or siHERC3 pool was measured by PM ELISA (n = 12 biological replicates). (G) Ubiquitination levels of HBH-∆F508-CFTR-3HA in CFBE Teton cells were measured by Neutravidin (NA) 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 = 4). (H) A schematic diagram of the HERC3 domain composition with the residue numbers at the domain boundaries. HERC3 mutants used in this study are also shown. (I) The effects of overexpressed Myc-HERC3 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 = 4). (J) The interaction of Myc-HERC3 variants with HBH-∆F508-CFTR-3HA in BHK cells was analyzed by NA pull-down and Western blotting. ∆F508-CFTR was rescued at 26°C incubation for 2 days, followed by a 1-h incubation at 37°C (A–D and F). Statistical significance was assessed by one-way ANOVA (A), or one-way repeated-measures (RM) ANOVA (B and I) with Dunnett’s multiple comparison tests, a two-tailed unpaired (C), or paired Student’s t test (G), or two-way ANOVA (E and F). 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 F1.

HERC3 participates in the ubiquitination and ERAD of ∆F508-CFTR. (A) The PM density of r∆F508-CFTR-HRP in CFBE Teton cells transfected with 50 nM siNT or siHERC3, as indicated (n = 9–12). Each independent experiment, consisting of three to four biological replicates (n), is color-coded. (B) Quantitative PCR analysis assessed HERC3 KD efficiency in CFBE Teton ∆F508-CFTR-HRP cells (n = 3). Each biological replicate (n) is color-coded: the averages from three technical replicates are shown in triangles. (C) The channel function of r∆F508-CFTR-3HA in CFBE Teton cells transfected with 50 nM siNT or siHERC3 pool was measured by YFP quenching assay. The initial YFP quenching rate was quantified as the CFTR function (right, n = 19). Each independent experiment, consisting of four to eight biological replicates (n), is color-coded. (D) Western blotting analyzed steady-state levels of ∆F508-CFTR-3HA with (r∆F508) or without 26°C rescue (∆F508) in CFBE Teton cells transfected with 50 nM siNT or siHERC3 pool. Na+/K+ ATPase (ATPase) was used as a loading control. B, immature form; C, mature form. Western blotting also confirmed HERC3 KD in CFBE Teton ΔF508-CFTR-3HA cells. Ponceau staining was used as a loading control. A filled triangle indicates HERC3. (E) Cellular ∆F508-CFTR-3HA stability in CFBE Teton cells transfected with 50 nM siNT or siHERC3 pool was measured by cell-based ELISA using an anti-HA antibody after CHX treatment (n = 12). (F) The PM stability of r∆F508-CFTR-3HA in CFBE cells transfected with 50 nM siNT, siRFFL pool, or siHERC3 pool was measured by PM ELISA (n = 12 biological replicates). (G) Ubiquitination levels of HBH-∆F508-CFTR-3HA in CFBE Teton cells were measured by Neutravidin (NA) 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 = 4). (H) A schematic diagram of the HERC3 domain composition with the residue numbers at the domain boundaries. HERC3 mutants used in this study are also shown. (I) The effects of overexpressed Myc-HERC3 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 = 4). (J) The interaction of Myc-HERC3 variants with HBH-∆F508-CFTR-3HA in BHK cells was analyzed by NA pull-down and Western blotting. ∆F508-CFTR was rescued at 26°C incubation for 2 days, followed by a 1-h incubation at 37°C (A–D and F). Statistical significance was assessed by one-way ANOVA (A), or one-way repeated-measures (RM) ANOVA (B and I) with Dunnett’s multiple comparison tests, a two-tailed unpaired (C), or paired Student’s t test (G), or two-way ANOVA (E and F). 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 F1.

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