Figure 2.
HERC3 and RNF5/185 additively reduce ∆F508-CFTR. (A and B) The cellular level of ∆F508-CFTR-3HA (A, n = 10) and PM level of r∆F508-CFTR-HRP (B, n = 8) in CFBE Teton cells transfected with 50 nM siRNA indicated was measured by cell-based ELISA using an anti-HA antibody and HRP assay, respectively. (C and D) The cellular level of ∆F508-CFTR-3HA (C, n = 15) and PM levels of r∆F508-CFTR-HRP induced by 26°C rescue (D, n = 8) in CFBE Teton cells transfected with 50 nM siRNA were measured by ELISA using an anti-HA antibody (C) and HRP assay (D), respectively. (E and F) Western blotting analyzed steady-state levels of r∆F508-CFTR-3HA in CFBE Teton cells transfected with 50 nM siRNA indicated (E). Ponceau staining was used as a loading control. B, immature form; C, mature form. The anti-RNF185 antibody detected both RNF5 and RNF185 because of the cross-reactivity. HERC3 KD was confirmed by quantitative PCR (F, n = 3). Each biological replicate (n) is color-coded: the averages from three technical replicates are shown in triangles. (G) The PM levels of r∆F508-CFTR-HRP induced by 3 µM VX-809 treatment at 37°C for 24 h in CFBE Teton cells transfected with 50 nM siRNA indicated (n = 8). (H) Representative traces (left) of the YFP fluorescence and quantification of the initial YFP quenching rate (right, n = 12) as a measure of rΔF508-CFTR function in CFBE cells transfected with 50 nM siRNA, as indicated. Each independent experiment consisting of 4 (B, D, and G), 5 (A and C), or 6 (H) biological replicates (n) is color-coded. Statistical significance was assessed by one-way RM ANOVA with Dunnett’s multiple comparison tests (F) or two-way ANOVA with Holm–Sidak multiple comparison tests, which revealed a significant main effect of HERC3 KD or RNF5/185 DKD, but no interaction between them (Pint > 0.05, C, D, and H) except for G (Pint = 0.012). Data distribution was assumed to be normal but was not formally tested. Data represent mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. Source data are available for this figure: SourceData F2.

HERC3 and RNF5/185 additively reduce ∆F508-CFTR. (A and B) The cellular level of ∆F508-CFTR-3HA (A, n = 10) and PM level of r∆F508-CFTR-HRP (B, n = 8) in CFBE Teton cells transfected with 50 nM siRNA indicated was measured by cell-based ELISA using an anti-HA antibody and HRP assay, respectively. (C and D) The cellular level of ∆F508-CFTR-3HA (C, n = 15) and PM levels of r∆F508-CFTR-HRP induced by 26°C rescue (D, n = 8) in CFBE Teton cells transfected with 50 nM siRNA were measured by ELISA using an anti-HA antibody (C) and HRP assay (D), respectively. (E and F) Western blotting analyzed steady-state levels of r∆F508-CFTR-3HA in CFBE Teton cells transfected with 50 nM siRNA indicated (E). Ponceau staining was used as a loading control. B, immature form; C, mature form. The anti-RNF185 antibody detected both RNF5 and RNF185 because of the cross-reactivity. HERC3 KD was confirmed by quantitative PCR (F, n = 3). Each biological replicate (n) is color-coded: the averages from three technical replicates are shown in triangles. (G) The PM levels of r∆F508-CFTR-HRP induced by 3 µM VX-809 treatment at 37°C for 24 h in CFBE Teton cells transfected with 50 nM siRNA indicated (n = 8). (H) Representative traces (left) of the YFP fluorescence and quantification of the initial YFP quenching rate (right, n = 12) as a measure of rΔF508-CFTR function in CFBE cells transfected with 50 nM siRNA, as indicated. Each independent experiment consisting of 4 (B, D, and G), 5 (A and C), or 6 (H) biological replicates (n) is color-coded. Statistical significance was assessed by one-way RM ANOVA with Dunnett’s multiple comparison tests (F) or two-way ANOVA with Holm–Sidak multiple comparison tests, which revealed a significant main effect of HERC3 KD or RNF5/185 DKD, but no interaction between them (Pint > 0.05, C, D, and H) except for G (Pint = 0.012). Data distribution was assumed to be normal but was not formally tested. Data represent mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. Source data are available for this figure: SourceData F2.

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