Fig 1: Protein processing and function of F312del-CFTR relative to WT-CFTR.(A) IB of protein lysates collected from HEK293 cells transiently transfected with F312del-CFTR, F508del-CFTR, WT-CFTR, and CAT-empty vector control. 40 µg of total cell lysates were electrophoresed and the IB probed with anti-CFTR antibody (596, Cystic Fibrosis Foundation Therapeutics). Anti-Na+K+ATPase (Abcam, ab76020) was used as loading control. Plots in lower panels show amount of mature CFTR protein relative to total CFTR protein normalized to the loading control using ImageJ (NIH) (n = 3 for each; 1-way ANOVA, ****P < 0.0001; ns, not significant). (B) Representative Isc recordings in CFBE41o– cells stably expressing either F312del-CFTR, WT-CFTR, or F508del-CFTR. We tested 2 different clones of F312del. After the baseline Isc stabilized, forskolin (10 µM, basolateral) and CFTRinh-172 (Inh172; 10 µM, apical) were sequentially and cumulatively added at the indicated times. Individual Isc recordings were acquired with Acquire and Analyze software (Physiologic Instruments) and plotted using GraphPad Prism 7.01 software. CFTR-specific function is defined as the difference (?Isc) between the sustained phase of the Isc response after stimulation with forskolin and the baseline achieved after adding CFTRinh-172; this value is used to calculate CFTR specific function generated by a variant as described previously (19) and in the Supplemental Material.