Figure 5. Co-translational misfolding and rescue of NBD1.

(A) After 5 min pre-warming the translation mix, we added ³⁵S-methionine and followed CFTR synthesis in the SP-cell system for 10–60 min. Analysis using 10% SDS-PAGE directly visualized CFTR nascent chain elongation with time. Full-length ΔF508 CFTR (<) first appeared after 30 min of translation, “⧫” indicates persistent unfinished nascent chains. (B) Wild-type and ΔF508 nascent chains were translated in vitro and harvested after 20, 30, or 60 min of synthesis. All nascent chains were subjected to increasing proteinase K concentrations and proteolytic fragments were separated by 12% SDS-PAGE. In the 5 µg/ml proteinase K treatment shown here, the NBD1-related 25 kDa fragment is marked by an arrowhead. The bracket indicates increased protease resistance of CFTR domains as a result of nascent chain elongation. (C) Similar experimental conditions as in B but with the I539T mutation in wild-type and ΔF508 CFTR background. Nascent chains were harvested after 30 min of synthesis and protease resistant fragments were quantified as in Figure 1B. Arrowhead indicates the NBD1-related 25 kDa fragment.