Biochemical analysis of corrector mechanism of action. (A) Effect of the indicated correctors (10 μM) on the expression pattern of ΔF508-CFTR-C_tHA in BHK cells. Cells were cultured for 24 hours at 37°C with or without correctors or at 27°C. Top: CFTR was visualized using anti-HA primary and HRP-conjugated secondary antibodies. Filled arrowhead, complex-glycosylated form (band C); open arrowhead, core-glycosylated form (band B). Bottom: Quantification of bands B and C (SEM; n = 4–5). (B) Effect of the indicated correctors (10 μM) on the expression pattern of ΔF508-CFTR in FRT epithelia. Confluent monolayers were treated as described in A, and immunoblot analysis was performed with the indicated primary antibodies. (C) Correlation between cell-surface density and PKA-activated chloride current. Cell-surface density of ΔF508-CFTR was determined using the radioactive anti-HA antibody binding assay and plotted against ΔF508-CFTR apical membrane currents in parallel experiments done on FRT cells. (D) Translational rate of ΔF508-CFTR was measured in the presence of correctors (10 μM) by monitoring [³⁵S]methionine/cysteine incorporation into CFTR during a 15-minute pulse labeling (SEM; n = 3). (E) Folding efficiency measured by pulse-chase assay. Top: The amount of newly synthesized ΔF508-CFTR was computed from radioactive incorporation during a 15-minute pulse (P). For measurement of folding efficiency, cells were pulsed for 150 minutes and than chased for 150 minutes (C). The amounts of core- (open arrowhead) and complex-glycosylated (filled arrowhead) forms were determined by phosphorimage analysis. Bottom: Maturation efficiency expressed as the percent of mature, complex-glycosylated ΔF508-CFTR relative to the newly synthesized pool, as shown in the top panel. (F) Stability of the core-glycosylated ΔF508-CFTR was measured following a 15-minute pulse labeling in the presence of the indicated correctors. Remaining radioactivity associated with CFTR was measured after 1 hour chase (SEM; n = 3). (G) Cell-surface stability of the rescued ΔF508-CFTR was measured by the anti-HA antibody binding assay before and after 2 hours chase in the presence of the indicated correctors. Data are expressed as the mean ± SEM of 3 independent experiments.