Figure 6.

Characterization of CF airway epithelial cells with antisense-mediated reduction of ClC-2. (A) Autoradiogram of genomic DNA from parental IB3-1 cells, and cell lines AS1, AS2, and AS3 that were transfected with the pBK antisense hClC-2 vector, digested with NsiI, and hybridized with a 0.5-kb ClC-2 cDNA probe. Clones AS1, AS2, and AS3 had intensely hybridizing fragments of 4.3 kb (arrow), consistent with the presence of multiple intact copies of the RSV promoter and antisense ClC-2 cDNA as shown in the map (Lower). Open arrow indicates endogenous ClC-2 genes. Solid boxes and arrows represent the RSV promoter and direction of transcription, and shaded boxes indicate the location of the cDNA probe relative to ClC-2. (B) Western blot of equal quantities (40 μg) of lysates from Parental and AS3 cells immunoblotted with chicken anti-rat ClC-2 antisera. Three bands were detected in the Parental cell lysate (first lane); two bands correspond to ClC-2 (85 and 89 kDa) and the third (80 kDa) is nonspecific (8). Both the 85 and 89 kDa forms of ClC-2 were severely reduced in the AS-3 cell line. Only the nonspecific band (80 kDa) was detected in the lysate of the AS3 cells (right lane). (C) I–V plots of mean currents ± SEM generated by Parental (open circles; n = 11) and AS-3 (open triangles; n = 11) cells in an acidic bath (pH 3.8) (Left). Right shows current values of individual cells at −160 mV (parental, open circles; AS3, solid circles) and mean current (circles with vertical bars, indicating SEM). The mean currents of the Parental and antisense cells differ significantly (P < 0.05) by anova and Bonferroni tests.