Figure 5.

Functional inhibition of CFTR leads to ubiquitination of CFTR at the PM of bronchial epithelial cells together with interaction with SQSTM1. (a and b) 16HBE14o- cells were treated with CFTR_inh172 (20 μM) with/without cystamine (250 μM) in the presence of CHX during the last 8 h of incubation. The incubation for 4 h at 4 °C was used to block endocytosis. (a) Left, expression of CHIP, SQSTM1 and Ubiquitin (Ab, P4D1) from membrane fraction. E-Cadherin, LAMP1, EEA1, Rab7 and β-actin were used as control of the membrane cell-surface protein fractionation. Right, densitometric analysis of protein levels expressed as fold increase. Mean±S.D., *P<0.05 compared with untreated cells, ^#P<0.01 versus CFTR_inh172-treated (analysis of variance (ANOVA)). (b) Protein samples from the PM were immunoprecipitated with anti-CFTR (clone CF3) and immunoblotted with anti-CFTR (clone M3A7), anti-SQSTM1 or anti-Ubiquitin (P4D1) Abs. Immunoprecipitation was performed with 500 μg of protein samples. Right, densitometric analysis shows the abundance of ubiquitylated CFTR adducts normalized for the expression level of their band C and expressed as fold increase relative to that of untreated cells (means±S.D. of three experiments). *P<0.05 compared with untreated cells, ^#P<0.01 versus CFTR_inh172-treated (ANOVA). (c) 16HBE14o- cells were treated with CFTR_inh172 in the presence or absence of cystamine. After 15 min following internalization, early endosome fraction was purified by sucrose cushion and immunoblotted for the early endosome marker EEA1. The absence of the late endosome marker Rab7 or lysosome marker LAMP-1 or membrane marker E-Cadherin confirms fraction-specific labeling. Left, representative immunoblot analysis of CFTR (clone 3MA7) and SQSTM1 in EEA1 positive fraction. Right, densitometric analysis of protein levels expressed as fold increase. Mean±S.D., *P<0.05 compared with untreated cells, ^#P<0.01 versus CFTR_inh172-treated (ANOVA). (d and e) 16HBE14o- cells were transfected with Flag-SQSTM1-wt or Flag-SQSTM1-deltaUBA with or without CFTR_inh-172. (d) Upper, immunoblots of Rab5 in whole cell lysates. Flag was used as control of transfection. β-actin expression was measured as a loading control. Bottom, densitometric analysis of protein levels expressed as fold increase. Mean±S.D., *P<0.05 compared with untreated cells (ANOVA). (e) The cells were treated with the proteasome inhibitor MG132 (50 μM). Upper, immunoblots of Rab5 in the detergent-insoluble fractions. Bottom, densitometric analysis of protein levels expressed as fold increase. Mean±S.D., *P<0.05 compared with untreated cells (ANOVA)