Figure 5.

GRINA Knockdown or Overexpression Inhibits Endosome-to-Golgi Retrieval

(A) Primary screen anti-CD8 antibody uptake images for control cells (top) and GRINA KD cells (bottom).

(B) Control (top) and GRINA KD (bottom) HeLa cells were stained for Golgi marker GM130 and Golgi glycoprotein-1 (GLG1).

(C) Cells stably expressing GFP-Rab6 were treated with GRINA siRNA (bottom) and compared to control cells (top) upon staining for TGN46 and VPS35.

(D) Quantitation by western blotting of lysates from control and GRINA-silenced HeLa cells and GFP-Rab6 cells.

(E–G) HeLa cells were transiently transfected with GRINA-Myc for 24 hr before fixing and staining. (E and F) GRINA-Myc colocalizes with both TGN46 and VPS35. In this example, GRINA-Myc expression (in the cell marked by an asterisk in E) reduced the cell’s TGN46 expression compared to surrounding untransfected cells and caused enlargement of VPS35-positive endosomes. The area in (E) magnified in (F) is indicated by a dashed line. (G) GRINA-Myc expression also perturbs CIMPR localization. In this example, GRINA-Myc transfection (in the cell marked by an asterisk) caused CIMPR to localize to round vesicular structures positive for GRINA-Myc, some of which appeared larger than regular endosomes, whereas TGN46 staining was almost absent in the transfected cell.

Scale bars in (A)–(C), (E), and (G), 20 μm, and in (F), 10 μm.