ABCMdb

PMID: 16131493

Swiatecka-Urban A, Brown A, Moreau-Marquis S, Renuka J, Coutermarsh B, Barnaby R, Karlson KH, Flotte TR, Fukuda M, Langford GM, Stanton BA
The short apical membrane half-life of rescued {Delta}F508-cystic fibrosis transmembrane conductance regulator (CFTR) results from accelerated endocytosis of {Delta}F508-CFTR in polarized human airway epithelial cells.
J Biol Chem. 2005 Nov 4;280(44):36762-72. Epub 2005 Aug 30., 2005-11-04 [PubMed]

Sentences

No. Mutations Sentence Comment

52 ABCC7 p.Tyr517Ala To construct the GFP-⌬F508-CFTR Y517A mutant, the GFP-⌬F508-CFTR cDNA sequence in pcDNA3.1 was mutated using the QuikChangeTM XL site-directed mutagenesis kit (Stratagene; La Jolla, CA). Login to comment 54 ABCC7 p.Tyr517Ala ABCC7 p.Tyr517Ala Transient transfection of the GFP-tagged CFTR cDNAs into parental CFBE41o-cells was performed using LipofectamineTM 2000 according to the manufacturer`s instructions. CFBE41o-cells grown on 40-mm tissue culture plates were incubated for 24 h with the transfection mixture (2 ␮g of cDNA and 4 ␮l of LipofectamineTM 2000 per plate) at 37 °C. Subsequently, cells were cultured in fresh medium at 27 °C for 36 h to increase the expression of GFP-⌬F508-CFTR and GFP-⌬F508-CFTR Y517A in the plasma membrane. Login to comment 64 ABCC7 p.Tyr517Ala ABCC7 p.Tyr517Ala Parental CFBE41o-cells transiently transfected with either GFP- ⌬F508-CFTR or GFP-⌬F508-CFTR Y517A were grown on plastic tissue culture plates. Login to comment 161 ABCC7 p.Tyr517Ala To test this hypothesis, we transiently expressed the GFP-tagged ⌬F508-CFTR or the GFP-⌬F508-CFTR Y517A mutant in parental CFBE41o-cells. Login to comment 164 ABCC7 p.Tyr517Ala The apical membrane expression of GFP-⌬F508-CFTR and GFP-⌬F508-CFTR Y517A was similar at steady state (Fig. 7A). Login to comment 165 ABCC7 p.Tyr517Ala Furthermore, as illustrated in Figs. 7, B and C, the apical membrane half-life of GFP-⌬F508-CFTR Y517A (1.0 h) did not differ from that of GFP-⌬F508-CFTR (1.1 h). Login to comment 194 ABCC7 p.Tyr517Ala FIGURE 7. Summary of experiments performed to determine the effects of the Y517A mutation in ⌬F508-CFTR on the apical membrane expression at steady state and the apical membrane half-life of rescued ⌬F508-CFTR. Login to comment 195 ABCC7 p.Tyr517Ala ABCC7 p.Tyr517Ala Parental CFBE41o-cells grown on plastic tissue culture plates were transiently transfected with either GFP-⌬F508-CFTR or the GFP-⌬F508-CFTR Y517A mutant. Login to comment 197 ABCC7 p.Tyr517Ala A, summary of biotinylation experiments demonstrating that the apical membrane expression of GFP-⌬F508-CFTR and GFP-⌬F508-CFTR Y517A at steady state did not differ. Login to comment 198 ABCC7 p.Tyr517Ala B, summary of biotinylation experiments demonstrating that the apical membrane half-life of GFP-⌬F508-CFTR (1.0 h) and GFP-⌬F508-CFTR Y517A (1.1 h) did not differ in CFBE41o-cells. Login to comment 199 ABCC7 p.Tyr517Ala Disappearance of GFP-⌬F508-CFTR and GFP-⌬F508-CFTR Y517A from the apical membrane was monitored over time in the presence of 20 ␮g/ml cyclohexamide (CHX) at 37 °C. Login to comment 201 ABCC7 p.Tyr517Ala "C,representativeWesternblotsdemonstratingthedisappearance of GFP-⌬F508-CFTR and GFP-⌬F508-CFTR Y517A from the apical membrane over time in CFBE41o-cells. Login to comment