Fig. 4

Mutations in LITAF result in loss-of-function, through a dominant-negative mechanism, rather than by toxic gain-of function. LITAF knockout (KO) Control, L125P and T115N patient fibroblasts were generated by CRISPR/Cas9. Antibiotic selection was used to maintain Cas9 and gRNA transduced cells, generating mixed populations of KO cells. a Western blotting was performed to confirm the loss of LITAF protein from mixed cell populations. Alpha-tubulin served as loading control. Full-length blots are shown in Additional file 2: Fig. 2. b Representative confocal microscopy images of fixed fibroblast lines that had been stained using an anti-LITAF antibody, to show the loss of LITAF expression from ~ 95% of cells. c Electron micrographs of control fibroblasts expressing Cas9, and following LITAF KO, and Cas9 expressing T115N fibroblasts, and following LITAF KO. LITAF KO cells display swollen endocytic compartments from either control backgrounds or T115N backgrounds