Figure 5. RPL12 depletion stabilizes CFTR interdomain assembly.

(A) WT-CFTR with domains annotated: TMD1 (dotted); NBD1 (black); regulatory domain (R) (white); TMD2 (light gray); NBD2 (dark gray). N, amino; C, carboxy. (B) CFBE expressing WT- or F508del-CFTR and treated with siRPL12 or NS siRNA were pulse labeled for 15 minutes and chased for 0–2 hours. RPL12 silencing was confirmed by immunoblotting (with tubulin-loading control). Lysates were digested with proteinase K and protease-resistant CFTR domain fragments immunoprecipitated/resolved by SDS-PAGE. Immature, ER-resident CFTR (band B, black arrowhead), fully glycosylated, mature CFTR (band C, white arrowhead), early domain fragments (T1a-c, N1a, T2a-b, N2a), and late domain fragments (T1d-f, T2b-c) are designated (see Methods). V, empty vector; asterisk indicates background. Brief chase applied here shows F508del-CFTR exits the ER slowly (i.e., band B at 2 hours remains significantly elevated following siRPL12 treatment), yet is stabilized in post-ER compartments, as evidenced by moderate band C accumulation (see also ref. 59). Increased proteolytic fragments predominantly reflect the ER-localized glycoform. (C) Quantification of RPL12 knockdown (n = 3–4). (D) Comparison of total radiolabeled CFTR (bands B and C) following RPL12 repression (n = 4–5). (E and F) Fold increase of domain-specific fragment intensities for WT- (E) or F508del-CFTR (F). Bands are corrected for total radiolabeling (n = 4–5). Note NS-treated F508del signal in the 2-hour chase is less than 2-fold over background in D and F. (C–F) Data are shown as mean ± SEM from siRPL12-treated cells normalized to NS siRNA (dotted line set to 100% or 1 as shown). Asterisks represent statistical comparison between siRPL12 and NS. Unequal variance t test on log₂-transformed data with post-hoc Bonferroni’s correction. α = 0.025 (C and D); α = 0.0125 (E and F). **P < 0.01; ***P < 0.001 (C and D). *P < 0.0125; **P < 0.001; ***P < 0.0001 (E and F).