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Published in final edited form as: Sci Signal. 2019 Jan 1;12(562):eaan7984. doi: 10.1126/scisignal.aan7984

Figure 3. — A. CX-4945 mediated inhibition of CK2α prevents phosphorylation of wild-type CFTR at Thr⁴²¹, Ser⁴²² and Ser⁴²⁷ as quantified with spiked-in synthetic, heavy isotope-labeled peptides. Data are means ± S.D. of n=3 experiments; statistical analysis noted below. B. Experimental outline for AHA labeling and enrichment of newly synthesized CFTR by click chemistry. C. Western blot of input lysate for CFTR-IP before click reaction. D. Western blot showing that wild-type CFTR maturation is dependent on CK2α phosphorylation in the RI element. Blots are representative of n=3 experiments. E. Quantification of newly synthesized immature (band B) and mature (band C) CFTR relative to time point 0 (h, hours). F. Western blot of mature CFTR (band C) in FRT cells expressing various CFTR mutants. G. Phosphorylation at Thr⁴²¹, Ser⁴²² and Ser⁴²⁷ in the various CFTR mutants described in (F) was quantified with spiked-in synthetic heavy isotope-labeled peptides, relative to the amount of mature CFTR present. Data are mean ± S.D. of ≥ n=4 independent measurements per site; statistical analysis noted below. (H to K) AHA pulse-chase assays in primary human bronchial epithelial (NHBE) cells (H) and FRT cells expressing G551D or wild-type CFTR (K) treated with CX-4945 or DMSO. Subsequent quantification of newly synthesized immature CFTR (band B) and mature CFTR (band C) at the time points indicated relative to time 0 (I and K). Top panels show input and loading control. SA-HRP, streptavidin-conjugated horse radish peroxidase. Time of AHA labeling before CFTR immunoprecipitation is indicated. Blots are representative, and the data are means ± S.D. of n=3 independent biological replicates. *P < 0.0332, **P < 0.0021, ***P < 0.0002, and ****P < 0.0001 by unpaired, two-tailed t- tests with Bonferroni-Sidak correction (A) or one-Way ANOVA with Tukey post-test (I and K).

Figure 3. — A. CX-4945 mediated inhibition of CK2α prevents phosphorylation of wild-type CFTR at Thr⁴²¹, Ser⁴²² and Ser⁴²⁷ as quantified with spiked-in synthetic, heavy isotope-labeled peptides. Data are means ± S.D. of n=3 experiments; statistical analysis noted below. B. Experimental outline for AHA labeling and enrichment of newly synthesized CFTR by click chemistry. C. Western blot of input lysate for CFTR-IP before click reaction. D. Western blot showing that wild-type CFTR maturation is dependent on CK2α phosphorylation in the RI element. Blots are representative of n=3 experiments. E. Quantification of newly synthesized immature (band B) and mature (band C) CFTR relative to time point 0 (h, hours). F. Western blot of mature CFTR (band C) in FRT cells expressing various CFTR mutants. G. Phosphorylation at Thr⁴²¹, Ser⁴²² and Ser⁴²⁷ in the various CFTR mutants described in (F) was quantified with spiked-in synthetic heavy isotope-labeled peptides, relative to the amount of mature CFTR present. Data are mean ± S.D. of ≥ n=4 independent measurements per site; statistical analysis noted below. (H to K) AHA pulse-chase assays in primary human bronchial epithelial (NHBE) cells (H) and FRT cells expressing G551D or wild-type CFTR (K) treated with CX-4945 or DMSO. Subsequent quantification of newly synthesized immature CFTR (band B) and mature CFTR (band C) at the time points indicated relative to time 0 (I and K). Top panels show input and loading control. SA-HRP, streptavidin-conjugated horse radish peroxidase. Time of AHA labeling before CFTR immunoprecipitation is indicated. Blots are representative, and the data are means ± S.D. of n=3 independent biological replicates. *P < 0.0332, **P < 0.0021, ***P < 0.0002, and ****P < 0.0001 by unpaired, two-tailed t- tests with Bonferroni-Sidak correction (A) or one-Way ANOVA with Tukey post-test (I and K).