Figure 2
From: Targeting DNAJB9, a novel ER luminal co-chaperone, to rescue ΔF508-CFTR
Knockdown DNAJB9 rescues both WT- and ΔF508-CFTR. (A–C) Knockdown of DNAJB9 by siRNA increased CFTR surface and functional expression in HEK293 cells. HEK293 cell lines stably expressing WT- or ΔF508-CFTR were transiently transfected with empty vector (Ø) or siRNA against DNAJB9 (siRNA) for 36–48 hours before assays. Parental cells that do not express CFTR were recruited as a negative control. All data were representative of three independent experiments. (A) WES analysis of CFTR and Vinculin (loading control). Total cell lysate was analyzed by WES system using anti-CFTR and anti-Vinculin antibodies. Each sample was analysed in triplicate. Student’s t-test was performed to determine the statistical significance. (**P < 0.01; ***P < 0.005). (B) In-Cell western to determine surface CFTR levels. Cells were reverse transfected by siRNA against DNAJB9 and RNF5 in 96-well plate. Forty-eight hours after transfection, cells were then fixed and surface CFTR was probed by anti-FLAG antibody followed by IRDye 800CW conjugated Goat anti-Rabbit 2nd antibody. CellTag 700 was used as cell quantity control. Samples without anti-FLAG antibody incubation were used as background control. Representative images were shown on the top and quantification was shown at the bottom. Each condition was done in triplicates. Student’s t-test was performed to determine the statistical significance. (*P < 0.05; **P < 0.01). (C) SPQ assay to assess CFTR function. Cells were seeded into 96-well plate 24 hours after transfection and then subjected to SPQ assay described in “Materials and Methods” section. A representative graph is shown for WT-CFTR (top) and ΔF508-CFTR (bottom). Each condition was performed in triplicate. (D) Knockdown DNAJB9 increased CFTR expression. Immunoblotting of mouse Jejunum membrane fraction from different genotypes, DNAJB9+/+ (WT), DNAJB9+/− (Het), and DNAJB9−/− (KO). CFTR and Na/K ATPase was probed using anti-CFTR and anti-Na/K ATPase antibodies, and Na/K ATPase was served as a membrane marker and loading control. Protein levels was quantified and ratio of KO/WT was determined. Full-length blots are presented in Fig. S11.
