Figure 3. Abnormal distribution of endocytic factors in Lowe syndrome fibroblasts.
(A and B) Immunofluorescence staining for OCRL in control (A) and Lowe syndrome fibroblasts (B), revealing the lack of OCRL in patient cells. Insets show higher magnifications of the boxed regions. Scale bar full size: 10 µm; inset: 1 µm. (C) Immunoblot analysis using a monoclonal antibody directed against the C-terminal region of human OCRL demonstrates the absence of the protein in patient cells. Actin is shown as a loading control. (D) High pressure liquid chromatography (HPLC) analysis of cell extracts showing an increase in PI(4,5)P2 levels in patient cells (Student's t test, *p = 0.01). (E–J) Immunofluorescence staining of control (left) and Lowe syndrome fibroblasts (right) for clathrin (E–F), AP-2 (G–H) and SNX9 (I–J) revealing the enhanced punctate immunoreactivity for endocytic factors in patient cells. Insets show higher magnifications of the boxed regions. Scale bar full size: 10 µm; inset: 1 µm. (K–L′) Rescue of the clathrin phenotype (K) and (L) in patient cells following expression of GFP-OCRLWT (K′) but not in cells expressing the catalytically inactive GFP-OCRLD523G (L′). Insets show higher magnifications of the boxed regions. Region and inset framed in white depict cells expressing the transfected construct (either GFP-OCRLWT (K–K′) or OCRLD523G (L–L′)). Region and inset framed in yellow show cells that did not express the GFP-tagged constructs. Scale bar full size: 10 µm; inset: 1 µm.