Figure 1.
Levels of 170 kD CFTR in control-PBMC treated with CI-2.
(A) Control-PBMC (1×106) were incubated in the absence or presence of the indicated concentrations of CI-2 for 24 hours. Total membrane fraction (10 µg of protein) was submitted to 6% SDS-PAGE followed by immunoblotting. Unfilled bars represent the quantification of mature 170 kD CFTR immunoreactive signals; a representative blot is shown in the inset. Filled bars represent intracellular calpain activity. The values reported are the arithmetic mean ± SD of three different quantifications performed on cells from three healthy donors. (B) Cells (1×107) untreated or treated with 2 µM CI-2 for 24 hours were fixed and CFTR localization was determined by confocal microscopy. Nuclei were stained with 2 µg/ml propidium iodide for 5 min [29]. CFTR signal (green fluorescence) was continuously monitored during cell scanning (from left to right following the white line) by using Laser Pix software. Each scanning trail is representative of 20 cells analyzed. (C) Control-PBMC (1×107) were incubated in the absence or presence of 2 µM CI-2 for 24 hours. Cell membranes recovered from 10% and 30% sucrose interface were used to detect CFTR by immunoblotting, to assay 5′-nucleotidase activity (NT), and to measure protein content (P). (D) Quantification and statistical analysis of 170 kD CFTR levels were carried out in PBMC from 16 healthy donors. PBMC were untreated or treated with 2 µM CI-2 for 24 hours. Statistical analysis with ANOVA test for a single factor revealed that the 95% confidence intervals of the different analysis did not superimpose showing a p-value≤0.0001.
Figure 2.
Levels of 170 kD F508del-CFTR in CF-PBMC treated with CI-2.
(A) CF-PBMC (1×106) were incubated in the absence or presence of the indicated concentration of CI-2 for 24 hours. Total membrane fraction (10 µg of protein) was submitted to 6% SDS-PAGE followed by immunoblotting. Unfilled bars represent the quantification of mature 170 kD CFTR immunoreactive signals; a representative blot is shown in the inset. Filled bars represent intracellular calpain activity. The values reported are the arithmetic mean ± SD of three different quantifications performed on cells of three patients. (B) Cells (1×107) untreated or treated with 2 µM CI-2 for 24 hours were fixed and CFTR localization was determined by confocal microscopy. Nuclei were stained with 2 µg/ml propidium iodide for 5 min [29]. CFTR signal (green fluorescence) was continuously monitored during cell scanning (from left to right following the white line) by using Laser Pix software. Each scanning trail is representative of 20 cells analyzed. (C) CF-PBMC (1×107) were incubated in the absence or presence of 2 µM CI-2 for 24 hours. Cell membranes recovered from 10% and 30% sucrose interface were used to detect CFTR by immunoblotting, to assay 5′-nucleotidase activity (NT), and to measure protein content (P). (D) Quantification and statistical analysis of 170 kD CFTR levels were carried out in PBMC from 28 patients. PBMC were untreated or treated with 2 µM CI-2 for 24 hours. Statistical analysis with ANOVA test for a single factor revealed that the 95% confidence intervals of the different analysis did not superimpose showing a p-value≤0.0001.
Figure 3.
Levels of CFTR activity in PBMC from controls and CF patients treated with CI-2.
(A) Control-PBMC and (B) CF-PBMC were treated in the absence or presence of 2 µM CI-2 for 24 hours. To detect specific CFTR activity cells were exposed to the indicated stimuli. The values are the arithmetic mean ± SD of three different evaluations performed on cells of five patients and correspond to the difference between the initial minus the final extracellular NaI nmoles measured in the conditions reported.
Figure 4.
Ratio between the level of CFTR activity and the intensity of the 170 kD protein band in PBMC from controls and CF patients treated with 2 µM CI-2.
CFTR activity was determined as described in Material and Methods. The levels of the mature 170 kD CFTR protein in control and CF-PBMC were obtained by scanning blots carried out as those shown in Figure 1 and 2. The values are the arithmetic mean ± SD of three different analyses.
Figure 5.
Levels of NHERF-1 in PBMC from controls and CF patients treated with CI-2.
PBMC (2.5×104), untreated or treated with 2 µM of CI-2 (CI-2) for 24 hours, obtained from 7 healthy donors (C) and 10 CF patients (CF), were submitted to 12% SDS-PAGE followed by immunoblotting. Native (50 kD) and digested (20 kD) NHERF-1 immunoreactive signals were quantified, and statistical analysis by ANOVA, followed by post hoc Tukey’s test, was carried out. Representative blot images for the two NHERF-1 forms are shown in the upper panel. *Significantly different from C (50 kD) p<0,05; **significantly different from CF (50 kD) p<0,001; ***significantly different from CF (20 kD) p<0,001.