Figure 5.

Expression and characterization of CFTR four-domain combinations. CFTR constructs lacking the indicated domain (ΔNBD1, ΔR, and ΔMSD2) (a) and ΔNBD2 (b) were expressed as M1 + RM2N2, M1N1 + M2N2, M1N1R + CD4T-N2, and M1N1RM2 (CFTR-1218X), respectively, in COS7 cells. Equal amounts of cell lysates were immunoblotted (top). Split wt CFTR was expressed as control, demonstrating the electrophoretic mobility shift of the complex-glycosylated R2M2N2 and M2N2 (dotted line) in the presence of complementing N-terminal fragments. The cell surface density of the M2 containing CFTR fragments, wt CFTR, and CD4T-N2 was measured by the immunoperoxidase assay (bottom). Means ± SEM, n = 3. (c) CFTR-1218X is complex-glycosylated. Cell lysates were treated with the endoglycosidase H or Endo F. Immunoblotting was performed with anti-HA Ab. (d) Maturation efficiency of CFTR-1218X was measured by metabolic pulse-chase experiments in stably transfected BHK cells. After a 10-min pulse labeling, cells were chased for 3 h, and CFTR was immunoprecipitated and visualized by fluorography. (e) Metabolic stability of the complex-glycosylated CFTR-1218X. Pulse-labeled BHK cells were chased as indicated, and labeled CFTR was visualized by fluorography and quantified by phosphorimage analysis by using the ImageQuant software (right). (f) In situ protease susceptibility of the wt and C-terminally truncated CFTR. Microsomes from wt, 1218X-, and 1158X CFTR-expressing BHK cells were subjected to limited proteolysis at the indicated trypsin concentration for 15 min on ice. Digestion patterns of CFTR were visualized by immunoblotting with MSD1-, NBD1-, and MSD2-specific anti-CFTR monoclonal Abs.