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. 2009 Jun 5;42(3):363–372. doi: 10.1165/rcmb.2008-0434OC

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Figure 2. — Ussing chamber analysis of ΔF508 and WT CFTR after low-temperature correction. (A and B) CFBE41o⁻ ΔF, (C and D) CFBE41o⁻ WT, and (E and F) CFBE41o⁻ parental cell monolayers were cultured at 27°C for 48 hours and studied in Ussing chambers at 37°C, as described in Materials and Methods. Under low chloride (1.2 mM NaCl, 115 mM Na gluconate), and 100 μM amiloride in the apical chamber, the basolateral membranes of the monolayers were permeabilized with amphotericin B (100 μM). After reaching baseline steady-state short-circuit currents (I_sc), the monolayers were treated sequentially with forskolin (FSK; 10 μM), genistein (GEN; 50 μM), and CFTR_inh-172 (three sequential additions: 20 + 20 + 60 μM). Representative tracings are shown on the left, and the summaries of the experiments are plotted on the right. Pretreatment of the cell monolayers during the last 1 hour at 27°C with corr-4a had no significant effect on the ΔI_SC compared with control (treated with 0.01% DMSO). The FSK responses of control ΔF508 CFTR–expressing monolayers were approximately 5% of the WT CFTR–expressing responses (B and D; [open bars], n = 11; 2.9 ± 0.46 versus 55.2 ± 5.12 mA/cm², respectively), whereas the FSK responses of the corr-4a–treated ΔF508 CFTR–expressing monolayers were 8% of the WT responses (B and D [gray bars]; n = 8–10; 3.66 ± 0.19 versus 48.49 ± 6.6 mA/cm², respectively). The CFBE41o⁻ parental cell line (E and F) did not respond to FSK treatment, but UTP (100 μM) produced a response, indicating that alternative channels had been activated (n = 4). Relative CFTR protein levels (G) and message levels (H) are shown for the CFBE41o⁻ WT, CFBE41o⁻ parental, CFBE41o⁻ ΔF, HeLa-ΔF, and HeLa parental cells. CFTR protein was immunoprecipitated from 500 μg total cellular proteins using 24-1 (an anti–C-terminal antibody), in vitro phosphorylated using γ³²P ATP, protein kinase A (PKA), separated on 6% SDS-PAGE, and detected using Phosphorimager analysis. CFTR mRNA levels are plotted relative to glyceraldehyde 3-phosphate dehydrogenase. CFTR mRNA levels in HeLa-ΔF and HeLa parental cells were measured as positive and negative controls, respectively. The results are given as means (±SD) (n = 4; * P < 0.005).

Figure 2. — Ussing chamber analysis of ΔF508 and WT CFTR after low-temperature correction. (A and B) CFBE41o⁻ ΔF, (C and D) CFBE41o⁻ WT, and (E and F) CFBE41o⁻ parental cell monolayers were cultured at 27°C for 48 hours and studied in Ussing chambers at 37°C, as described in Materials and Methods. Under low chloride (1.2 mM NaCl, 115 mM Na gluconate), and 100 μM amiloride in the apical chamber, the basolateral membranes of the monolayers were permeabilized with amphotericin B (100 μM). After reaching baseline steady-state short-circuit currents (I_sc), the monolayers were treated sequentially with forskolin (FSK; 10 μM), genistein (GEN; 50 μM), and CFTR_inh-172 (three sequential additions: 20 + 20 + 60 μM). Representative tracings are shown on the left, and the summaries of the experiments are plotted on the right. Pretreatment of the cell monolayers during the last 1 hour at 27°C with corr-4a had no significant effect on the ΔI_SC compared with control (treated with 0.01% DMSO). The FSK responses of control ΔF508 CFTR–expressing monolayers were approximately 5% of the WT CFTR–expressing responses (B and D; [open bars], n = 11; 2.9 ± 0.46 versus 55.2 ± 5.12 mA/cm², respectively), whereas the FSK responses of the corr-4a–treated ΔF508 CFTR–expressing monolayers were 8% of the WT responses (B and D [gray bars]; n = 8–10; 3.66 ± 0.19 versus 48.49 ± 6.6 mA/cm², respectively). The CFBE41o⁻ parental cell line (E and F) did not respond to FSK treatment, but UTP (100 μM) produced a response, indicating that alternative channels had been activated (n = 4). Relative CFTR protein levels (G) and message levels (H) are shown for the CFBE41o⁻ WT, CFBE41o⁻ parental, CFBE41o⁻ ΔF, HeLa-ΔF, and HeLa parental cells. CFTR protein was immunoprecipitated from 500 μg total cellular proteins using 24-1 (an anti–C-terminal antibody), in vitro phosphorylated using γ³²P ATP, protein kinase A (PKA), separated on 6% SDS-PAGE, and detected using Phosphorimager analysis. CFTR mRNA levels are plotted relative to glyceraldehyde 3-phosphate dehydrogenase. CFTR mRNA levels in HeLa-ΔF and HeLa parental cells were measured as positive and negative controls, respectively. The results are given as means (±SD) (n = 4; * P < 0.005).