PMC

Apparent FRET efficiencies, E, obtained after acceptor photobleaching of HEK293T cells co-transfected with vectors encoding ECFP-ΔF508-CFTR and αβγ-ENaC (in αβγ-ENaC, one each of the three subunits tagged at the C terminus with EYFP). Also, E is shown for control constructs. Error bars, S.D. of at least five cells imaged.

A, β-ENaC and ΔF508-CFTR co-IP signal does not occur postlysis. The lysates of cells expressing β-ENaC were mixed with the lysates of cells expressing ΔF508-CFTR; immunoprecipitation was carried out with a C-terminal CFTR antibody, followed by blotting with β-ENaC subunit-specific antibody. B, ΔF508-CFTR does not interact with the Cl channel ECFP-ClC1 following co-expression in HEK-293T cells. An anti-GFP monoclonal Ab JL-8 (BD Living Colors), which recognizes ECFP, was used to blot for ECFP-ClC-1. No co-IP signal was detected in IP of cells co-expressing the ΔF508-CFTR and ClC-1 (bottom panel, far right lane).

Apparent FRET efficiencies, E, obtained after acceptor photobleaching of HEK293T cells co-transfected with vectors encoding ECFP-ΔF508-CFTR and αβγ-ENaC (in αβγ-ENaC, one each of the three subunits tagged at the C terminus with EYFP) following the partial correction by glafenine (A), low temperature (B), and combination of Corr3a and Corr4a (C). Also, E is shown for control constructs. Errors bars, S.D. of at least five cells imaged.

Apparent FRET efficiencies, E, obtained following fluorescence lifetime measurements of HEK293T cells co-transfected with vectors encoding ECFP-ClC-1 and αβγ-ENaC (in αβγ-ENaC, one each of the three subunits tagged at the C terminus with EYFP) with no treatment (A) and following partial correction by low temperature (B) and glafenine (C). Not shown are the results of the partial correction with Corr3a and Corr4a. Also, E is shown for control constructs. Error bars, are S.D. of at least three cells imaged.

Western blot analysis of tagged CFTR constructs. A, the blot was carried out using a polyclonal Ab raised against the NBD2 domain of CFTR and probed with an anti-rabbit HRP using chemiluminescence. ENaC subunits interact with both WT and ΔF508 CFTR. α-ENaC (B), β-ENaC (C), and γ-ENaC (D) could be co-immunoprecipitated using a C-terminal CFTR antibody when co-expressed with ΔF508-CFTR; αβγ-ENaC and ΔF508-CFTR were co-expressed, and then individual subunits were probed. No ENaC signal was detected when α-, β, or γ-ENaC or ΔF508-CFTR was expressed alone. The left panels show that α-, β, or γ-ENaC can only be detected when expressed in HEK-293 cells. Therefore, ENaC subunits are not endogenously expressed in those cells. These experiments were repeated at least three times, and similar results were obtained.

Evaluation of the changes in surface expression following low temperature rescue of ΔF508-CFTR. A combination of surface biotinylation and co-IP was utilized to monitor changes in surface expression. The cells were co-transfected with ΔF508-CFTR and all three ENaC subunits; one of three subunits in αβγ-ENaC was tagged with EYFP - α-EYFP-βγ (A), β-EYFP-αγ (B), and γ-EYFP-αβ (C). The low temperature rescue enhanced the co-IP signal and was statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; corresponding densitometry bar graphs). The changes in densitometry value at 27 °C were normalized to that at 37 °C (100%). The bands at 56 kDa in each panel indicate the high molecular weight immunoglobulin band. These experiments were repeated at least three times with similar results. Error bars, S.E.